Systems and methods for growing cells in vitro

ABSTRACT

A system for growing cells comprising a bioreactor chamber for growing the cells, a delivery system delivering a perfusion solution to the bioreactor chamber for perfusion of the perfusion solution through the cells, a dialysis system having a dialyzer, a dialysate for performing a dialysis and a filter for reducing ammonia content in said dialysate, and a controller that circulates the perfusion solution through the dialyzer and the dialysate through the filter.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/316,667, entitled “Systems and Methods for Growing Cells In Vitro,”filed Jan. 10, 2019, which is a 371 of PCT Application Serial No.PCT/IL2017/050790, entitled “Systems and Methods for Growing Cells InVitro,” filed Jul. 11, 2017, which claims the benefit of priority ofU.S. Provisional Patent Application Ser. No. 62/360,495, entitled“Systems and Methods for Growing Cells In Vitro,” filed on Jul. 11,2016. Each of these applications is incorporated herein by reference inits entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 76441_ST25.txt, created on Jan. 10, 2019,comprising 29,718,555 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference. The sequencelisting submitted herewith is identical to the sequence listing formingpart of the international application.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to cellgrowth and, more particularly, but not exclusively, to a system and amethod for growing cells in vitro.

The current world population is over 7 billion and still rapidlygrowing. In order to support the nutritional requirement of this growingpopulation, increasing amount of land is dedicated for food production.The natural sources are insufficient to fulfill the demand. This has ledto famine in some parts of the world. In other parts of the world theproblem is being addressed by large-scale production of animals in densefactory farms under harsh conditions. This large-scale production is notonly causing great suffering to animals, but in addition, organoarseniccompounds and antibiotics are used to increase food efficiency andcontrol infection, increasing arsenic levels and drug-resistancebacteria in meat products. It can also increase the number of diseasesand the consequences thereof for both animals and humans. Large scaleslaughtering is currently required to fulfill the current foodrequirements and as a consequence of large-scale disease outbreaks suchas the occurrence of porcine pestivirus and mad cows disease. Thesediseases also result in loss of the meat for human consumption thuscompletely denying the purpose for which the animals were being bred inthe first place. In addition the large-scale production is reducing theflavor of the finished product. A preference exists among those that canafford it for non-battery laid eggs and non-battery produced meat. Notonly it is a matter of taste but also a healthier choice therebyavoiding consumption of various feed additives such as growth hormones.Another problem associated with mass animal production is theenvironmental problem caused by the vast amounts of fecal mater theanimals produce and which the environment subsequently has to deal with.Also the large amount of land currently required for animal productionor the production of feed for the animals which cannot be used foralternative purposes such as growth of other crop, housing, recreation,wild nature and forests.

Several approaches have been disclosed to address these problems.

U.S. Pat. No. 685,390 discloses a non-human tissue engineered meatproduct and a method for producing same. The meat product comprisesmuscle cells that are grown ex-vivo and is used for food consumption.The muscle cells may be grown and attached to a support structure andmay be derived from any non-human cells. The meat product may alsocomprise other cells such as fat cells or cartilage cells, or both, thatare grown ex-vivo together with the muscle cells.

U.S. Pat. No. 7,270,829 discloses a meat product containing in-vitroproduced animal cells in a three dimensional form and a method forproducing the meat product. The method comprises the culturing in-vitroof animal cells in medium free of hazardous substances for humans on anindustrial scale thereby providing three dimensional animal tissuesuited for human consumption, wherein the cells are muscle cells, somitecells or stem cells.

U.S. Pat. No. 8,703,216 discloses methods and engineered meat productsformed as a plurality of at least partially fused layers, wherein eachlayer comprises at least partially fused multicellular bodies comprisingnon-human myocytes and wherein the engineered meat is comestible, andwherein the non-human myocytes are adhered and/or cohered to oneanother; and the multicellular bodies are arranged adjacently on anutrient-permeable support substrate and maintained in culture to allowthe multicellular bodies to at least partially fuse to form asubstantially planar layer for use in formation of engineered meat.

U.S. Patent application US2011/0091604 discloses examples of methods,systems and computer accessible mediums related to producing syntheticmeat, with a substrate configured to support cell growth, which can beseeded with cells. The seeded substrate may be rolled through abioreactor having a roll-to-roll mechanism, thereby allowing nutrientsand growth factors to interact with the cells. The seeded substrate maybe stretched to simulate muscle action. The seeded substrate may bemonitored for uniformity of cell growth as it is rolled through thebioreactor. A film of synthetic meat is obtained from the substrate.

U.S. Patent application US2011/0301249 discloses methods for producingin-vitro cultured protein products that are enhanced with stem cells,providing nutrients to an animal by feeding the animal with the in-vitrocultured protein products.

WO 2015/066377 discloses methods for enhancing cultured meat production,such as livestock-autonomous meat production, wherein the meat can beany metazoan tissue or cell-derived comestible product intended for useas a comestible food or nutritional component by humans, companionanimals, domesticated or captive animals whose carcasses are intendedfor comestible use, service animals, conserved animal species, animalsused for experimental purposes, or cell cultures.

U.S. Pat. No. 8,802,361 discloses a perfusion solution comprisingspecific metabolic agents, antioxidant agents, and membrane stabilizeragents that can help improve preservation, organ viability, and in somecases recover organs that would otherwise being unusable fortransplantation, wherein the perfusion solution can be used incombination with hypothermic machine perfusion. It has been found thatcombination of the perfusion solution and hypothermic machine perfusioncan help prevent or reduce further damage to the organ and restore theorgan's anti-oxidant system, stabilize the cellular cytoskeleton andcellular membranes, inhibit arachidonic acid pathway, provide oncoticsupport, reduce interstitial edema formation, and help restore energystores within the organ.

One of the main problems of the aforementioned techniques is therelation between cost, time and quality of the product, with a long timeto produce, at extremely high costs with a mediocre quality that cannotand will not replace the current meat derived from livestock.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a system for growing cells, the system comprising:

a bioreactor chamber for growing the cells;

a delivery system configured to deliver a perfusion solution to thebioreactor chamber for perfusion of the perfusion solution through thecells at a perfusion rate;

a dialysis system having a dialyzer and a dialysate for performing adialysis and a filter for reducing ammonia content in the dialysate; and

a controller configured to circulate the perfusion solution out of thebioreactor chamber through the dialyzer and back into the bioreactorchamber, and to circulate the dialysate out of the dialyzer through thefilter and back into the dialyzer.

According to an aspect of some embodiments of the present inventionthere is provided a method of growing cells, the method comprising:

growing the cells in a bioreactor chamber;

delivering a perfusion solution to the bioreactor chamber for perfusionof the perfusion solution through the cells;

circulating the perfusion solution out of the bioreactor chamber througha dialyzer having a dialyzer therein and back into the bioreactorchamber; and

circulating the dialysate out of the dialyzer, through a filter selectedfor reducing ammonia content in the dialysate, and back into thedialyzer.

According to an aspect of some embodiments of the present inventionthere is provided a system for growing cells, the system comprising:

a bioreactor chamber for growing the cells;

a delivery system configured to deliver a perfusion solution to thebioreactor chamber for perfusion of the perfusion solution through thecells at a perfusion rate;

a dialysis system having a dialyzer for performing a dialysis; and

a controller configured to increase the perfusion rate with time, and tocirculate the perfusion solution out of the bioreactor chamber,separately through the dialyzer and the delivery system, and back intothe bioreactor chamber;

wherein at least 90% of a volume of the perfusion solution that exitsthe bioreactor chamber is circulated back into the bioreactor chamberduring an entire growth period of the cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of growing cells, the method comprising:

growing the cells in a bioreactor chamber;

delivering by a delivery system a perfusion solution to the bioreactorchamber for perfusion of the perfusion solution through the cells at aperfusion rate that increases with time; and

circulating the perfusion solution out of the bioreactor chamberseparately through a dialyzer and the delivery system, and back into thebioreactor chamber;

wherein at least 90% of a volume of the perfusion solution that exitsthe bioreactor chamber is circulated back into the bioreactor chamberduring an entire growth period of the cells.

According to an aspect of some embodiments of the present inventionthere is provided a system for growing a suspension cell culture, thesystem comprising:

a bioreactor chamber for growing the suspension cell culture;

a delivery system configured to deliver a perfusion solution to thebioreactor chamber for perfusion of the perfusion solution through thesuspension cell culture at a perfusion rate;

a dialysis system having a dialyzer for performing a dialysis; and

a controller configured to circulate the perfusion solution out of thebioreactor chamber through the dialyzer and back into the bioreactorchamber, while maintaining at least 95% of cells forming the suspensioncell culture in the bioreactor chamber during the circulation.

According to an aspect of some embodiments of the present inventionthere is provided a method of growing a suspension cell culture, themethod comprising:

growing the suspension cell culture in a bioreactor chamber;

delivering a perfusion solution to the bioreactor chamber for perfusionof the perfusion solution through the suspension cell culture; and

circulating the perfusion solution out of the bioreactor chamber througha dialyzer and back into the bioreactor chamber, while maintaining atleast 95% of cells forming the suspension cell culture in the bioreactorchamber during the circulation.

According to an aspect of some embodiments of the present inventionthere is provided an adipocyte obtainable according to the methods ofsome embodiments of the invention.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a cultured fat on a proteinmatrix, comprising generating the adipocyte cell from the fibroblastaccording to the method of some embodiments of the invention, whereinthe culturing is performed on a plant-derived protein matrix, therebygenerating the cultured fat on the protein matrix.

According to an aspect of some embodiments of the present inventionthere is provided an in-vitro method of generating an adipocyte cellfrom a fibroblast, comprising culturing a spontaneously immortalizedfibroblast in a serum-free medium comprising oleic acid and a PPAR-gammaagonist or activator, thereby generating the adipocyte cell.

According to an aspect of some embodiments of the present inventionthere is provided a cultured fat in a plant-derived protein matrix.

According to an aspect of some embodiments of the present inventionthere is provided an in-vitro method of generating a myocyte from afibroblast, comprising upregulating expression within a spontaneouslyimmortalized fibroblast of a polypeptide selected from the groupconsisting of myoD1 and myogenin.

According to an aspect of some embodiments of the present inventionthere is provided a myocyte obtainable according to the methods of anyone of claims 57-62.

According to an aspect of some embodiments of the present inventionthere is provided an in-vitro method of screening for a small moleculecapable of producing a myocyte, comprising:

(a) transfecting a spontaneously immortalized fibroblast with a nucleicacid construct comprising a nucleic acid sequence encoding a reporterpolypeptide under a transcriptional control of a promoter specificallyactive in myocytes,

(b) contacting a transfected fibroblast resultant of step (a) with atleast one small molecule of a plurality of small molecules, and

(c) detecting activity of the reporter polypeptide above apre-determined threshold in the transfected fibroblast following step(b), wherein presence of the activity above the pre-determined thresholdis indicative that the at least one small molecule is capable ofconverting the spontaneously immortalized fibroblast into the myocyte.

According to an aspect of some embodiments of the present inventionthere is provided an in-vitro method of generating an edible meat,comprising culturing:

(a) a spontaneously immortalized fibroblast in a serum-free medium underconditions suitable for converting the fibroblast into an adipocyte,and/or

(b) a spontaneously immortalized fibroblast in a serum-free medium underconditions suitable for converting the fibroblast into a myocyte,thereby generating the edible meat.

According to an aspect of some embodiments of the present inventionthere is provided an in-vitro method of generating an edible meat,comprising culturing:

(a) a spontaneously immortalized fibroblast in a serum-free medium underconditions suitable for converting the fibroblast into an adipocyte,and/or

(b) a spontaneously immortalized fibroblast in a serum-free medium underconditions suitable for converting the fibroblast into a myocyte,

(c) an endothelial cell,

thereby generating the edible meat.

According to an aspect of some embodiments of the present inventionthere is provided an edible meat obtainable from the method of any oneof claims 67-81.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a spontaneously immortalizedfibroblast, comprising:

(a) culturing avian embryo cells in the presence of a serum-containingmedium under adherent culture conditions to thereby obtain chickenembryonic fibroblasts,

(b) passaging the avian embryonic fibroblasts for at least 10-12passages in the serum-containing medium under the adherent conditionsuntil culture collapse, wherein the culture collapse is characterized bysenescence and/or death of at least 90% of the avian embryonicfibroblasts,

(c) isolating at least one colony which survived the culture collapse inthe serum-containing medium for at least additional 20 passages,

thereby generating the spontaneously immortalized fibroblast.

According to an aspect of some embodiments of the present inventionthere is provided a spontaneously immortalized chicken fibroblastobtainable by the method of some embodiments of the invention.

According to some embodiments of the invention, at least 90% of a volumeof the perfusion solution that exits the bioreactor chamber iscirculated back into the bioreactor chamber during an entire growthperiod of the cells

According to some embodiments of the invention, the cells form a tissue.

According to some embodiments of the invention, the cells form acultured meat product.

According to some embodiments of the invention, the dialyzer comprises afilter selected to reduce ammonia content of the perfusion solution.

According to some embodiments of the invention, the perfusion rateincreases over time.

According to some embodiments of the invention, the increment isexponential.

According to some embodiments of the invention, there is a plurality ofbioreactor chambers, all being in fluid communication with the samedialyzer, and wherein the dialyzer applies the dialysis to perfusionsolutions circulated out of each of the bioreactor chambers.

According to some embodiments of the invention, the dialyzer isconfigured to ensure that at least one protein exiting the bioreactorchamber with the perfusion solution is circulated back into thebioreactor chamber.

According to some embodiments of the invention, the at least one proteinis albumin.

According to some embodiments of the invention, there is from about 0.1liters to about 10 liters of the perfusion solution per one kilogram ofcells in the bioreactor chamber.

According to some embodiments of the invention, there is from about 0.1liters to about one liter of the perfusion solution per one kilogram ofcells in the bioreactor chamber.

According to some embodiments of the invention, the delivery of theperfusion solution is via a fluidic circuit constituted to enrich theperfusion solution by a culture medium and oxygen.

According to some embodiments of the invention, the fluidic circuit isconstituted to enrich the perfusion solution also by carbon dioxide.

According to some embodiments of the invention, the fluidic circuit isconstituted to trap or remove bubbles present in the perfusion solution.

According to some embodiments of the invention, the fluidic circuit isconstituted to heat the perfusion solution.

According to some embodiments of the invention, the delivery and thecirculation is without discarding the perfusion solution throughout thecell growth.

According to some embodiments of the invention, the cells form acultured meat product and wherein the bioreactor chamber is at most 5liters in volume.

According to some embodiments of the invention, the bioreactor chamberis at most 5 liters in volume.

According to some embodiments of the invention, the fibroblast is anavian fibroblast.

According to some embodiments of the invention, the avian is selectedfrom the group consisting of: chicken, duck, goose, and quail.

According to some embodiments of the invention, the fibroblast is achicken embryonic fibroblast.

According to some embodiments of the invention, the spontaneouslyimmortalized fibroblast is non-genetically modified.

According to some embodiments of the invention, the PPAR-gamma agonistor activator is a small molecule.

According to some embodiments of the invention, the small molecule isselected from the group consisting of Thiazolidinedione,3-Isobutyl-1-methylxanthine (IBMX), phenamil, GW7845, RG14620, andHarmine.

According to some embodiments of the invention, the small molecule isrosiglitazone.

According to some embodiments of the invention, the serum-free medium isdevoid of animal contaminants.

According to some embodiments of the invention, the serum-free medium isdevoid of human contaminants.

According to some embodiments of the invention, the serum-free mediumcomprises insulin or a substitute thereof, and basic fibroblast growthfactor (bFGF) or a substitute thereof, and at least one additional agentselected from the group consisting of dexamethasone, transferrin,selenium, EGF or a substitute thereof, and PGE2.

According to some embodiments of the invention, the substitute of theinsulin comprises IGF-1 or a stabilized Long R3 IGF-1

According to some embodiments of the invention, the substitute of theEGF comprises an EGF-R agonist.

According to some embodiments of the invention, the EGF-R agonistcomprises NSC-228155 at a concentration of 5-50 ng/ml.

According to some embodiments of the invention, the substitute of thebFGF is a small molecule or a synthetic agonist of the FGF-signalingpathway.

According to some embodiments of the invention, the synthetic agonist isC19-jun at a concentration of 10-20 ng/ml.

According to some embodiments of the invention, the dexamethasone isprovided at a concentration range of 0.01 nM-10 μM.

According to some embodiments of the invention, the bFGF is provided ata concentration range of 0.1-30 ng/ml.

According to some embodiments of the invention, the EGF is provided at aconcentration range of 0.1-30 ng/ml.

According to some embodiments of the invention, the PGE2 is provided ata concentration range of 0.01 nM-10 μM.

According to some embodiments of the invention, the plant-derivedprotein matrix is from the legume (Fabaceae) family, from the cerealfamily or from the pseudocereal family.

According to some embodiments of the invention, the plant-derivedprotein matrix comprises a soy protein or a pea protein.

According to some embodiments of the invention, the cultured fat of someembodiments of the invention is obtainable by the method of someembodiments of the invention.

According to some embodiments of the invention, the upregulation is ofthe myoD1 and myogenin polypeptides.

According to some embodiments of the invention, the chicken myoD1polypeptide is encoded by a polynucleotide comprising the nucleic acidsequence set forth by SEQ ID NO:5.

According to some embodiments of the invention, the chicken myogeninpolypeptide is encoded by a polynucleotide comprising the nucleic acidsequence set forth by SEQ ID NO:7.

According to some embodiments of the invention, the chicken myoD1polypeptide is encoded by the nucleic acid construct set forth by SEQ IDNO: 1 or 3.

According to some embodiments of the invention, the chicken myogeninpolypeptide is encoded by the nucleic acid construct set forth by SEQ IDNO: 2.

According to some embodiments of the invention, the serum-free mediumcomprises oleic acid and a PPAR-gamma agonist.

According to some embodiments of the invention, the endothelial cell isa spontaneously immortalized endothelial cell.

According to some embodiments of the invention, the endothelial cell isnon-genetically modified.

According to some embodiments of the invention, step (a) and step (b)are effected simultaneously in the same culture system.

According to some embodiments of the invention, step (a) and step (b)are effected in two distinct culture systems.

According to some embodiments of the invention, steps (a), (b) and (c)are effected simultaneously in the same culture system.

According to some embodiments of the invention, the culturing isperformed on a scaffold.

According to some embodiments of the invention, the culturing isperformed in a perfusion system.

According to some embodiments of the invention, the culturing isperformed on an edible hollow fiber cartridge.

According to some embodiments of the invention, the culturing isperformed on a vegetable-derived matrix.

According to some embodiments of the invention, the vegetable-derivedmatrix is from a cereal family, legume (Fabaceae) family or apseudocereal family.

According to some embodiments of the invention, the legume is soy orpea.

According to some embodiments of the invention, the culturing isperformed in a suspension culture devoid of substrate adherence.

According to some embodiments of the invention, the culturing isperformed in the system of some embodiments of the invention.

According to some embodiments of the invention, the edible meat of someembodiments of the invention is in a form of a patty or nugget with adensity of about 200×10⁶ cells/gram.

According to some embodiments of the invention, the serum-containingmedium is a DMEM/F12 based medium.

According to some embodiments of the invention, the serum in the mediumcomprises about 15% fetal bovine serum (FBS).

According to some embodiments of the invention, the chicken embryo isobtained from a fertilized broiler chicken egg grown for 10-12 days.

According to some embodiments of the invention, the spontaneouslyimmortalized chicken fibroblast of some embodiments of the inventionbeing capable of a continuous passaging for at least 30 passages.

According to some embodiments of the invention, the spontaneouslyimmortalized chicken fibroblast of some embodiments of the inventionbeing capable of at least 90 population doublings.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

Implementation of the method and/or system of embodiments of theinvention can involve performing or completing selected tasks manually,automatically, or a combination thereof. Moreover, according to actualinstrumentation and equipment of embodiments of the method and/or systemof the invention, several selected tasks could be implemented byhardware, by software or by firmware or by a combination thereof usingan operating system.

For example, hardware for performing selected tasks according toembodiments of the invention could be implemented as a chip or acircuit. As software, selected tasks according to embodiments of theinvention could be implemented as a plurality of software instructionsbeing executed by a computer using any suitable operating system. In anexemplary embodiment of the invention, one or more tasks according toexemplary embodiments of method and/or system as described herein areperformed by a data processor, such as a computing platform forexecuting a plurality of instructions. Optionally, the data processorincludes a volatile memory for storing instructions and/or data and/or anon-volatile storage, for example, a magnetic hard-disk and/or removablemedia, for storing instructions and/or data. Optionally, a networkconnection is provided as well. A display and/or a user input devicesuch as a keyboard or mouse are optionally provided as well.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

FIGS. 1A and 1B are schematic illustrations of a system suitable forgrowing cells according to some embodiments of the present invention.

FIGS. 2A-E demonstrate the derivation of a spontaneously immortalizedline of chicken embryonic fibroblasts. FIG. 2A—Broiler chicken embryoextracted from fertilized egg on day 11. FIG. 2B—morphology of primarychicken embryonic fibroblasts (CEF) after 1 population doubling (“PD1”). FIG. 2C—Morphology of spontaneously immortalized chickenfibroblasts (CSIF) post crisis (after 90 population doublings (“PD 90”).FIG. 2D—Growth kinetics of CSIF cultured in 15% serum (FBS, blackcurve), serum-free medium as described in Example 5 of the Examplessection which follows (“SFM”, red curve) and commercially availableTheraPEAK™ (LONZA WALKERSVILLE, INC. Walkersville, Md., 217930127)medium (“T-PEAK”, green curve). FIG. 2E—Doubling time of CSIF culturedin 15% serum (FBS, black column), serum-free medium as described inExample 6 of the Examples section which follows (“SFM”, red column) andcommercially available TheraPEAK™ (LONZA WALKERSVILLE, INC.Walkersville, Md., 217930127) medium (“T-PEAK”, green column). Note thatthe immortalized chicken fibroblast cell line (CSIF) exhibit the samegrowth kinetics and doubling time in the presence of serum-free mediumformulation uncovered by the present inventor (as described in Example 6of the Examples section which follows) when compared to theserum-containing medium. Also note that the commercially availableTheraPEAK™ (LONZA WALKERSVILLE, INC. Walkersville, Md., 217930127)failed to support the expansion of the CSIF cells (FIG. 2D), and thecells cultured therein exhibit an elongated doubling time of 40 hours ascompared to less than 20 hours in either the serum-containing medium ofthe SFM of some embodiments of the invention.

FIGS. 3A-F depict the development and identification of serum-freemedium for CSIF propagation. Shown are sulforhodamine B stain (FIGS.3A-E) and protein content quantification (FIG. 3F) following 72 hours ofculture with 15% serum (“FBS”), minimal serum-free medium (MIN) alone(FIG. 3B) or with 10 ng/ml basic Fibroblast Growth Factor (bFGF, FIG.3C), 5 ng/ml Epidermal Growth Factor (EGF, FIG. 3D), 0.01 μMProstaglandin E2 (PGE2, FIG. 3E), or 10 ng/ml Growth Hormone. Serum-freemedium (SFM) contained MIN medium supplemented with bFGF (10 ng/ml), EGF(5 ng/ml), and PGE2 (0.01 M). “MIN” medium included: DMEM/F12, 0.1 μMdexamethasone, insulin, transferrin, and selenium (ITS), 12 μM linoleicand oleic acids, and L-Analyl-L-Glutamine. (GlutaMAX); Note that thecells cultured in the SFM exhibit a similar cell mass (as determined byprotein content) as the cells cultured in a medium supplemented with 15%FBS.

FIGS. 4A-D depict conversion of CSIF to adipocytes in serum-free medium.FIGS. 4A-C. LipidTOX™ (Thermo Fisher Scientific) neutral lipid stain ofserum-free cultures of CSIF exposed to either 400 μM oleic acid (OA)alone (FIG. 4A) or with 0.5 mM IBMX (OA+IBMX, FIG. 4B), or 10 μMRosiglitazone (OA+TZD, FIG. 4C) for 7 days. Both small molecules showstrong adipogenesis in the presence of OA. FIG. 4D—Normalizedintracellular lipid content (in arbitrary fluorescent units) of CSIFcultures treated for 4 and 7 days as prescribed above. 400 μM OA withsmall molecules IBMX or TZD show optimal results.

FIGS. 5A-E depict conversion of CSIF to myocytes. FIG. 5A—Phase image ofCSIF expressing Dox-inducible MyoD1 and Myogenin (MYOG) for 6 days(“d6”). FIGS. 5B-C—CSIF expressing rat myosin light chain COP-GFPreporter (rMLC3-GFP) following Dox-induced MyoD1+MYOG expression for 11days (FIG. 5B) or 30 days (FIG. 5C). About 2-4% of the cultures becomepositive for MLC3 (Green). MLC3 positive myoblasts maintain elongatedfiber morphology for over 30 days in vitro (FIG. 5C). FIG.5D—Fluorescence staining using phalloidin (F-Actin probe, green) showingmultinucleated cells (syncytia) as well as some striation following 7days in culture. Nuclei are stained with Hoechst (blue). FIG.5E—Immunofluorescence staining for α1-skeletal muscle actin (ACTA1,green) and Troponin T (red) showing a clear muscle phenotype by day 7 ofinduction. Nuclei are stained with Hoechst (blue).

FIG. 6 is a schematic illustration of the pinducer-VP64-cMyoD1 nucleicacid construct used to express chicken MyoD1 in a spontaneouslyimmortalized fibroblast under Dox-induction. Shown are the “centralpolypurine tract/central termination sequence” (CPPT/CTS) element (inorange), which creates a “DNA flap” that increases nuclear importationof the viral genome during target cell infection and improves vectorintegration and transduction efficiency); the tetracycline responseelement (in pink); the VP64 transcriptional activator (in peach); the HAepitope tag (in yellow); and the cMYOD1 coding sequence (in light blue).

FIG. 7 is a schematic illustration of the pInducer20-cMyogenin nucleicacid construct used to express the chicken myogenin in a spontaneouslyimmortalized fibroblast under Dox-induction. Shown are the “centralpolypurine tract/central termination sequence” (CPPT/CTS) element (inlight peach), the tetracycline response element (in Turquoise); theminimal CMV promoter (white arrow head); and the cMyogenin codingsequence (in light blue).

FIG. 8 is a schematic illustration of the rat MLC3 enhancer-promoter inpGreenFire lentiviral vector used to show the conversion of aspontaneously immortalized fibroblast into a myocyte. Shown are thecentral polypurine tract” (CPPTS) element (in yellow); the rat MLC3enhancer (in light blue); the rat MLC3 promoter (in orange) and the COPGFP coding sequence (in green). It is noted that this vector can be usedto screen for small molecules capable of converting a spontaneouslyimmortalized fibroblast into a myocyte.

FIG. 9 is a schematic illustration of a system suitable for growingcells as designed in a prototype design, according to some embodimentsof the present invention.

FIGS. 10A and 10B are graph showing the produce mass and appliedperfusion rates (FIG. 10A), and accumulated glucose consumption (FIG.10B), as obtained in experiments performed according to some embodimentsof the present invention.

FIGS. 11A-C depict tissue formation and vascularization. FIG.11A—Sulforhodamine B stain of 3D collagen scaffolds loaded with 150×10⁶spontaneously immortalized chicken fibroblasts (CSIF) and 15×10⁶spontaneously immortalized rat microvascular endothelial cells (RCEC)per millimeter of volume. FIG. 11B—Phase image of 3D scaffolds loadedwith high density of CSIF and RCEC co-culture following 11 days ofperfusion in a bioreactor. FIG. 11C—Confocal cross-section of 3Dscaffolds loaded with RCEC (red label) and iPS-derived cells (green)showing vascular network formation and close cell-cell interactionsfollowing 11 days of perfusion in a bioreactor.

FIG. 12 a schematic illustration of the pInducer20-cMyoD1 nucleic acidconstruct used to express the chicken MyoD1 in a spontaneouslyimmortalized fibroblast under Dox-induction. Shown are the “centralpolypurine tract/central termination sequence” (CPPT/CTS) element (inlight peach), the tetracycline response element (in Turquoise); theminimal CMV promoter (white arrow head); and the cMyoD1 coding sequence(in orange).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to cellgrowth and, more particularly, but not exclusively, to a system and amethod for growing cells in vitro.

The present inventor has described a system for culturing cells whichcan be used, in some embodiments of the present invention, forgenerating edible meat.

Chicken meat has been a major source of dietary protein since the dawnof the agricultural revolution. Production has traditionally been local,with families and later small villages growing their own grain-fedanimals. However, rapid urbanization and population growth driven by theindustrial revolution led to the development of intensive farmingmethods. Factory farms now produce close to 9 billion chickens each yearin the United States, with animal growth and transportation producing18% of current greenhouse emissions. It was recognized by the presentinventor that large amount of chicken meat (e.g., over 70% in the UnitedStates) contains unsafe levels of arsenic, and antibiotic resistantbacteria. It was also recognized by the present inventor thattransportation and animal density lead to widespread fecal contaminationof chicken meat leading to increased salmonella infection.

Laboratory-grown meat allows growing meat from animal cells understerile conditions. It was find by the present inventor that it ispossible to produce a sufficient amount of cells per unit mass of meatproduct (e.g., from about 500 to about 200×10⁶ cells per gram), withoutthe use of animal products, such as fetal bovine serum. However, whilemany cell culture techniques have been developed over the past 50 yearsfor biological research, the present inventor found that such culturetechniques are incredibly wasteful, requiring a large volume of culturemedium to produce a small mass of laboratory-grown meat. For example,known techniques require a volume of about 230 liter of to produce about1 Kg of meat, translating to a cost of at least $4,600 per Kg due tomedium costs alone.

For purposes of better understanding some embodiments of the presentinvention, the construction and operation of industrial scale cellmanufacturing techniques will be described.

Known in the art are several industrial scale cell manufacturingtechniques. These include a 10,000 liter fed-batch process, and a 1,000liter concentrated perfusion process. Typical media cost at currentprices is estimated at about $20 per liter L for fed-batch processes andabout $5 per liter for concentrated perfusion processes. For ideal CHOcells, the fed-batch processes allow achieving cell densities of about25×10⁶ cells/ml, and the concentrated perfusion processes allowachieving cell densities of about 100×10⁶ cells/ml. These cell densitiesmean that a 10,000 liter fed batch reactor can produce 1,250 kg massevery 19 days, while a 1,000 liter perfusion reactor can produce 500 kgmass every 30 days. The fed batch process consumes 12,500 liter mediumincluding the seed train, while the perfusion process consumes 2,120liters medium. These numbers translate to $200 per kg mass for fed batchprocess and $21 per kg mass for perfusion process for the culture mediumcosts alone.

It is recognized that consumable costs are often less than a third ofthe cost of good. One parameter is the capital costs. 10,000 liter fedbatch facilities are known to cost of $50 million or more, and 1,000perfusion facilities are known to cost $30 million or more. Assuming aliberal 10% annual depreciation and maintenance costs, an industrialscale 10,000 fed batch facility can produce 24,000 kg per year at anannual maintenance cost of about $5,000,000, resulting in a capital costof $200 per kg mass produced. An industrial scale 1,000 perfusionfacility can produce 6,000 kg per year at an annual maintenance cost ofabout $3,000,000, resulting in a capital cost of about $500 per kg massproduced.

The present inventor devised a cell growing technique that outperformsthese conventional processes.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details of construction and the arrangement of thecomponents and/or methods set forth in the following description and/orillustrated in the drawings and/or the Examples. The invention iscapable of other embodiments or of being practiced or carried out invarious ways.

FIGS. 1A and 1B are schematic illustrations of a system 1000 suitablefor growing cells 302 according to some embodiments of the presentinvention. System 1000 can be used form growing many types of cells. Insome embodiments of the present invention cells 302 form a suspensionculture, useful, for example, for cellular therapy or for protein orvaccine production, in some embodiments of the present invention cells302 form a tissue, useful, for example, for tissue transplantation, andin some embodiments of the present invention cells form a cultured meat.

System 1000 preferably comprises a bioreactor chamber 300 for growingthe cells 302 therein, a delivery system 100 configured to deliver aperfusion solution to bioreactor chamber 300 for perfusion of theperfusion solution through the cells, and a dialysis system 200 having adialyzer 20 and a dialysate 22 for performing a dialysis to exchangenutrient and byproduct. Bioreactor chamber 300 can employ any techniquefor growing cells, including, without limitation, a hollow fibercartridge, a packed bed, or a vascularized embedded tissueconfiguration. The perfusion of the perfusion solution through the cellsis optionally and preferably continuous. In some embodiments of thepresent invention, at the end of the growth cycle there are from about0.1 liters to about 10 liters, e.g., 1 liter, of perfusion solution perone kilogram of cells in the bioreactor chamber. Bioreactor chamber 300can have any size but in preferred embodiments of the present inventionbioreactor chamber 300 is at most 5 liters, e.g., from about 1 liter toabout 5 liters, in volume. These embodiments are particularly usefulwhen system 1000 comprises a plurality of bioreactor chambers as furtherdetailed hereinbelow. Bioreactor chamber 300 can typically facilitategrowth of muscle tissue, from an initial amount of about 20 mg to aconsumable amount of at least 500 grams, e.g., 1000 grams or more.

System 1000 preferably operates in a generally closed loop fluidic mode,wherein the perfusion solution exits bioreactor chamber 300 through oneor more outlet ports 308 into delivery system 100 and dialysis system200, treated in these systems and then returns back into bioreactorchamber 300 through one or more inlet ports 306. The operation isreferred to as “a close loop operation” in the sense that the perfusionsolution itself is not discarded. Thus, system 1000 is optionally andpreferably devoid of any mechanism for removing the perfusion solutionout of system 1000 into an external waste removal device, and devoid ofany mechanism that increases the amount of perfusion solution during theoperation. It is to be understood, however, that the contents of theperfusion solution are changed during operation by interacting withsystems 100 and 200. In various exemplary embodiments of the inventionwherein at least 90% or at least 92% or at least 94% or at least 96% orat least 98% of a volume of the perfusion solution that exits bioreactorchamber 300 (either to system 100 or to system 200) is circulated backinto bioreactor chamber 300 at all times over a period of at least 4days or at least 5 days or at least 6 days or at least 7 days or atleast 8 days or at least 9 days or at least 10 days or at least 12 daysor at least 14 days or at least 16 days or at least 18 days or at least20 days, or during the entire growth cycle of cells 302.

System 1000 optionally and preferably comprises a controller 304 forcontrolling delivery system 100, dialysis system 200 and/or bioreactorchamber 300. Controller 304 optionally and preferably comprises acircuit configured for performing the various operations describedherein. In some embodiments of the present invention controller 304 is acomputerized controller. Representative control lines from controllerare shown as dotted lines. One of ordinary skills in the art, providedwith the details described herein would know how to construct controllines between controller 304 and other controllable components of system1000.

In some embodiments of the present invention controller 304 circulatesthe perfusion solution out of bioreactor chamber 300 through dialyzer 20and back into bioreactor chamber 300. This can be achieved by means of apump 21 which is controlled by controller 304. In various exemplaryembodiments of the invention the circulation is executed whilemaintaining at least 95% or at least 96% or at least 97% of the cells302 in bioreactor chamber 300 during the circulation. Preferably,dialyzer 20 is configured to ensure that at least one protein (e.g.,albumin), more preferably all proteins, exiting bioreactor chamber 300with the perfusion solution is circulated back into bioreactor chamber300. This can be done for example, by providing a membrane dialyzer witha membrane that ensures that the respective protein (such as, but notlimited to, albumin, with a molecular weight of about 66.5 kDa) iscirculated back to bioreactor chamber 300 without entering the dialysate22 of dialysis system 200. The advantage of this embodiment is thatalbumin is a carrier protein of growth factors, hormones, and fattyacids, and can therefore facilitate growth of cells 302 for at least aperiod that equals its characteristic half-life (about 20 days). Thissignificantly reduces the production cost of the cells since albumin,hormones, and growth factors are the main cost driver of culture media.

In various exemplary embodiments of the invention a filter is employedin dialysis system 200 to remove ammonia from the portion of theperfusion solution that enters dialysis system 200. This can be achievedby providing dialysis system 200 with a filter 24 selected for reducingammonia content in dialysate 22. In these embodiments, controller 304optionally and preferably circulates dialysate 22 out of dialyzer 20through filter 24 and back into dialyzer 22, for example, by controllinga pump 23 in dialysis system 200. Ammonia is a product of peptidedegradation and glutamine breakdown. Ammonia become toxic and limitscell growth when it reaches 5 mM concentration. The close loop operationof the present embodiments preferably separates the protein-containingmedium from the protein-free dialysate that can be scrubbed of ammoniawithout losing protein to non-specific absorption. Suitable for thepresent embodiments are filters such as, but not limited to, packedZeolites particles or carbon meshes. Zeolite-based oxygen concentratorsystems are widely used to produce medical-grade oxygen. The zeolite isused as a molecular sieve to create purified oxygen from air using itsability to trap impurities, in a process involving the adsorption ofnitrogen, leaving highly purified oxygen and clearing ammonia from thesolution.

System 1000 preferably operates in cycles, wherein the cell growth isinitiated at the beginning of the cycle, and the grown cells are takenout of the chamber to provide a cellular product (suspension culture,tissue, cultured meat) at the end of the cycle. Typically, a 10 daycycle is employed but other cycle durations are also contemplated. Insome embodiments of the present invention controller 304 ensures thatthe perfusion rate within bioreactor chamber 300 increases over timeduring the operation cycle. Preferably, the increment is exponential.The increment of the perfusion rate need not to be continuous, albeit acontinuous increment of the perfusion rate is also contemplated. Forexample, the perfusion rate can be increased intermittently at certaindays during the operation cycle. Typically, but not necessarily, thefirst increment is effected several days after the beginning of thecycle. At the end of the cycle, the perfusion rate is preferably atleast 20 ml/s or at least 25 ml/s or at least 30 ml/s or at least 35ml/s, e.g., 36 ml/s or more.

Referring again to FIGS. 1A-B, the delivery of the perfusion solution isoptionally and preferably via a fluidic circuit 102, which is optionallyand preferably controlled by controller 304, for example, by means of apump 11 in delivery system 100, and is constituted to enrich theperfusion solution by a culture medium and one or more gaseous media,such as, but not limited to, oxygen, carbon dioxide and nitrogen. Thisis optionally and preferably achieved by means of a culture mediumreservoir 10 that enriches the perfusion solution by the culture medium,and a mass transfer device 12 such as, but not limited to, an oxygenatoror the like, that enriches the perfusion solution by one or more gaseousmedia. Typically, mass transfer device 12 provides a mixture of Oxygenfrom about 21% to about 95%, Carbon dioxide from about 0% to about 10%and balanced to 100% by Nitrogen. Preferably, mass transfer device 12maintains a generally constant (e.g., with 10% or less tolerance) pH. Ina representative example, which is not to be considered as limiting,mass transfer device 12 provides a mixture of about 80% Oxygen about 5%Carbon dioxide and about 15% Nitrogen.

Optionally, fluidic circuit 102 is constituted to trap or remove bubblespresent in the perfusion solution. This can be achieved by means of abubble handling device 12 that may include a bubble trap and/or adebubbler. The advantage of trapping or removing the bubbles is thatbubbles that inadvertently introduced into the bioreactor chamber 300can negatively affect the operation of system 1000 since bubbles arecytotoxic to cells and can potentially rupture their cell membranes, andso trapping or removing the bubbles can improves the performance ofsystem 1000. In some embodiments of the present invention fluidiccircuit 102 is also constituted to heat the perfusion solution,optionally and preferably before the perfusion solution enters thebioreactor chamber 300.

System 1000 can comprise more than one bioreactor chamber 300. Thispreferred embodiment is illustrated in FIG. 1B. Shown are severalbioreactor chambers, each being optionally and preferably similar tobioreactor chamber 300 as described herein, and several deliverysystems, each being optionally and preferably similar to delivery system100 as described herein, wherein each delivery system circulates, forexample, by means of pump 11, the perfusion solution through one of thebioreactor chambers.

A portion of the perfusion solution also exits the bioreactor chambersfor dialysis as further detailed hereinabove. In the illustratedembodiments, which is not to be considered as limiting, portions of theperfusion solutions from all the bioreactor chambers enter a maincirculation channel 1002 circulating the perfusion solutions into thesame dialysis system, which is optionally and preferably similar todialysis system 200 as described herein. Thus, in these embodiments,System 1000 comprises a plurality of bioreactor chambers, a respectiveplurality of delivery systems, and a shared dialysis system which applydialysis to perfusion solutions of all the bioreactor chambers.

The bioreactor chambers, delivery systems and dialysis system areoptionally and preferably controlled by controller 304 as furtherdetailed hereinabove.

The number of bioreactor chambers (and of respective delivery systems)in system 1000 is preferably selected such that the aggregate volumes ofthe perfusion solutions in the bioreactor chambers can be dialyzed bythe dialysis system. Typically, there are from about 10 to about 500,e.g., about 100 bioreactor chambers in system 1000. For example, whenthe dialysis system is constructed to dialyze about V liters ofperfusion solution, and each of the bioreactor chambers has about vliters of perfusion solution, the number of bioreactor chambers insystem 1000 is V/v. As a representative example which is not to beconsidered as limiting, V can be about 500 and v can be about 5, so thatV/v is about 100. The advantage of having a plurality of relativelysmall bioreactor chambers is that it allows having a relatively highperfusion rate.

Following is a more detailed description of system 1000, according tosome embodiments of the present invention.

Dialyzer 20 can be, for example, hollow fiber dialyzer, such as, but notlimited to, the hollow fiber dialyzer that is commercially distributedby Rancho Spectrum Labs (Rancho Dominguez, Calif.). The dialyzer caninclude membrane having an area of from about 500 to about 1000, e.g.,about 790 cm², and a molecular weight cutoff of from about 20 to about40, e.g., about 30 kDa. A fraction of the perfusion solution can bediverted using a pump 21, such as, but not limited to, a peristalticpump, to system 200 through dialyzer 20. The dialyzer 20 dialyzes theperfusion solution by counter-flow exposure to a protein-free dialysate22, recirculated through a filter 24, such as, but not limited to, anammonia filter 24, an additional pump 23, such as, but not limited to, aperistaltic pump. Temperature within system 1000 is optionally andpreferably maintained at a physiological range selected based on thetype of animal cell 302 being grown. For chicken, for example, thetemperature can be from about 38 to about 42° C., for beef cow thetemperature can be from about 36.7 to about 39° C., for pig thetemperature can be from about 38 to about 39° C., etc.

Bioreactor Chamber

In some embodiments of the present invention, the chamber 300 has avolume and internal dimensions that are configured and arranged toreceive the growing cells and retain the cells within its volume while asufficient amount of perfusion solution continuously circulates throughthe growing cells. The chamber 300 is optionally and preferablyspecifically adapted to the type of cells in it, in order to provide theadequate environment for the cells to grow and minimize mechanicaldamage or physical stress that can block vascular supply of oxygen andnutrients to the growing cells.

Peristaltic Pumps

Peristaltic pumps are known in the art. In order to provide any personskilled in the art with the required information to perform the presentinvention a brief explanation will be provided. A peristaltic pump is atype of positive displacement pump used for pumping a variety of fluids(www.en.dot.wikipedia.dot.org/wiki/Peristaltc_pump, incorporated hereinas reference). The fluid is contained within a flexible tube fittedinside a circular pump casing (though linear peristaltic pumps have beenmade). A rotor with a number of “rollers”, “shoes”, “wipers”, or “lobes”attached to the external circumference of the rotor compresses theflexible tube. As the rotor turns, the part of the tube undercompression is pinched closed (or “occludes”) thus forcing the fluid tobe pumped to move through the tube. Additionally, as the tube opens toits natural state after the passing of the cam (“restitution” or“resilience”) fluid flow is induced to the pump. This process is calledperistalsis and is used in many biological systems such as thegastrointestinal tract. Typically, there will be two or more rollers, orwipers, occluding the tube, trapping between them a body of fluid. Thebody of fluid is then transported, at ambient pressure, toward the pumpoutlet. Peristaltic pumps may run continuously, or they may be indexedthrough partial revolutions to deliver smaller amounts of fluid.

Peristaltic pumps are typically used to pump clean/sterile or aggressivefluids because cross contamination with exposed pump components cannotoccur. Some common applications include pumping IV fluids through aninfusion device, aggressive chemicals, high solids slurries and othermaterials where isolation of the product from the environment, and theenvironment from the product, are critical. It is also used inheart-lung machines to circulate blood during a bypass surgery as thepump does not cause significant hemolysis.

Peristaltic pumps are also used in a wide variety of industrialapplications. Their unique design makes them especially suited topumping abrasives and viscous fluids.

The minimum gap between the roller and the housing determines themaximum squeeze applied on the tubing. The amount of squeeze applied tothe tubing affects pumping performance and the tube life—more squeezingdecreases the tubing life dramatically, while less squeezing can causethe pumped medium to slip back, especially in high pressure pumping, anddecreases the efficiency of the pump dramatically and the high velocityof the slip back typically causes premature failure of the hose.Therefore, this amount of squeeze becomes an important design parameter.

The term “occlusion” is used to measure the amount of squeeze. It iseither expressed as a percentage of twice the wall thickness, or as anabsolute amount of the wall that is squeezed.

Let y denote an occlusion, g denote minimum gap between the roller andthe housing, and t denote wall thickness of the tubing. Then y=2t−g,when expressed as the absolute amount of squeeze, and y=(2t−g)/(2t)×100,when expressed as a percentage of twice the wall thickness. Theocclusion is typically 10 to 20%, with a higher occlusion for a softertube material and a lower occlusion for a harder tube material.

Thus for a given pump, the most critical tubing dimension becomes thewall thickness. An interesting point here is that the inside diameter ofthe tubing is not an important design parameter for the suitability ofthe tubing for the pump. Therefore, it is common for more than one ID beused with a pump, as long as the wall thickness remains the same.

Inside diameter: for a given rpm of the pump, a tube with larger insidediameter (ID) will give higher flow rate than one with a smaller insidediameter. Intuitively the flow rate is a function of the cross sectionarea of the tube bore.

The flow rate in a peristaltic pump is determined by many factors, suchas the tube internal diameter (ID), where higher flow rate are obtainedwith larger ID, the pump head's outer diameter (OD), where higher floware obtained with larger OD, and the pump head's rotation speed, wherehigher flow rate are obtained with higher rotation speed. It isrecognized that increasing the number of rollers typically does notincrease the flow rate. Rather it typically decreases the flow rate byreducing the effective (fluid-pumping) circumference of the head.Increasing rollers typically decreases the amplitude of the fluidpulsing at the outlet by increasing the frequency of the pulsed flow.

The length of tube (measured from initial pinch point near the inlet tothe final release point near the outlet) does not affect the flow rate.However, a longer tube implies more pinch points between inlet andoutlet, increasing the pressure that the pump can generate.

The present embodiments contemplate any of several variations ofperistaltic pumps. Hose pumps can typically operate against up to 16 barin continuous service, use shoes (rollers only used on low pressuretypes) and have casings filled with lubricant to prevent abrasion of theexterior of the pump tube and to aid in the dissipation of heat, and usereinforced tubes, often called “hoses”. This class of pump is oftencalled a “hose pump”. The advantage with the hose pumps over the rollerpumps is the high operating pressure of up to 16 bar. With rollers maxpressure can arrive up to 12 Bar. Tube pumps are typically lowerpressure peristaltic pumps having dry casings and use rollers along withnon-reinforced, extruded tubing. This class of pump is sometimes calleda “tube pump” or “tubing pump”. These pumps employ rollers to squeezethe tube. Except for the 360° eccentric pump design as described below,these pumps have a minimum of 2 rollers 180° apart, and may have as manyas 8, or even 12 rollers. Increasing the number of rollers increase thepressure pulse frequency of the pumped fluid at the outlet, therebydecreasing the amplitude of pulsing.

The present embodiments contemplate any of several variations of rollerdesigns. In a fixed occlusion pump, the rollers have a fixed locus as itturns, keeping the occlusion constant as it squeezes the tube. Inspring-loaded rollers, the rollers in this pump are mounted on a spring.This design helps overcome the variations in the tube wall thicknessover a broader range. Regardless of the variations, the roller impartsthe same amount of stress on the tubing that is proportional to thespring constant, making this a constant stress operation. The spring isselected to overcome not only the hoop strength of the tubing, but alsothe pressure of the pumped fluid.

The operating pressure of these pumps is determined by the tubing and bythe motor's ability to overcome the hoop strength of the tubing and thefluid pressure.

While the embodiments above are described with a particular emphasis toperistaltic pumps, it is to be understood that other pumps, such as, butnot limited to, positive displacement pumps, impulse pumps, velocitypumps, gravity pumps, steam pumps and valveless pumps, can be employed.

Medium Perfusate

Depending on the type of cell source grown in the device of the presentinvention a specific medium perfusate is used.

Cell culture medium often contains fetal bovine serum (FBS) thatprovides attachment factors, fatty acids, growth factors, hormones, andalbumin. FBS can usually be replaced with serum replacement (e.g.KO-serum) that is composed of amino acids, vitamins, and trace elementsin addition to transferrin, insulin, and lipid-rich bovine serumalbumin. While both transferrin and insulin are produced in bacteriausing recombinant technology, albumin is usually animal derived.However, plant and bacteria-derived recombinant human albumin (e.g.Cellastim™) are available through several companies, includingSigma-Aldrich (St. Louis, Mo.).

Chicken embryonic fibroblast (CEF) medium is traditionally composed ofM199 or DMEM/F12 medium supplemented with 15% FBS, and glutamine.However, serum-free medium for the growth of mammalian fibroblasts isnow readily available. Medium is for mammalian cells (e.g. cow, pig) iscomposed of M199 supplemented with 0.5 mg/mL albumin, 0.6 μM linoleicacid, 0.6 μg/mL lecithin, 5 ng/mL bFGF, 5 ng/mL EGF, 30 pg/mL TGFβ1, 7.5mM glutamine, 1 μg/mL hydrocortisone, 50 μg/mL ascorbic acid, and 5μg/mL insulin. This medium PCS-201-040 is available from ATCC (Manassas,Va.) and is reported to support 4-fold faster proliferation of humanfibroblasts. Under some conditions, insulin could be replaced withIGF-1, or the stabilized Long R3 IGF-1 (Sigma). EGF can be replaced withthe EGF-R agonist NSC-228155 (Sakanyan et al. Sci. Reports. 2014). FGFcan similarly be replaced with a small molecule or synthetic agonistsuch as C19-jun (Ballinger et al. Nature. Biotech. 1999). Chickenhepatocytes are similarly supported by a serum-free culture mediumdesigned for human and mouse hepatocytes. Medium is composed of WilliamsE basal medium supplemented with albumin, insulin, transferrin, andhydrocortisone (1).

Oxygenator

An oxygenator is a medical device that is capable of exchanging oxygenand carbon dioxide in the blood of human patient during surgicalprocedures that may necessitate the interruption or cessation of bloodflow in the body, a critical organ or great blood vessel. These organscan be the heart, lungs or liver, while the great vessels can be theaorta, pulmonary artery, pulmonary veins or vena cava. An oxygenator istypically utilized by a perfusionist in cardiac surgery in conjunctionwith the heart-lung machine. However, oxygenators can also be utilizedin extracorporeal membrane oxygenation in neonatal intensive care unitsby nurses (www.en.dot.wikipedia.dot.org/wiki/Oxygenator, incorporatedhereinafter as reference).

For most cardiac operations such as coronary artery bypass grafting, thecardiopulmonary bypass is performed using a heart-lung machine (orcardiopulmonary bypass machine). The heart-lung machine serves toreplace the work of the heart during the open bypass surgery. Themachine replaces both the heart's pumping action and the lungs' gasexchange function. Since the heart is stopped during the operation, thispermits the surgeon to operate on a bloodless, stationary heart.

One component of the heart-lung machine is the oxygenator. Theoxygenator component serves as the lung, and is designed to expose theblood or perfusion medium to oxygen and remove carbon dioxide. It isdisposable and contains about 2-4 m² of a membrane permeable to gas butimpermeable to blood, in the form of hollow fibers. Blood flows on theoutside of the hollow fibers, while oxygen flows in the oppositedirection on the inside of the fibers. As the blood passes through theoxygenator, the blood comes into intimate contact with the fine surfacesof the device itself. Gas containing oxygen and medical air is deliveredto the interface between the blood and the device, permitting the bloodcells to absorb oxygen molecules directly.

In some embodiments of the present invention, an oxygenator is providedas mass transfer device 12 to exchange of gases in the medium used togrow the cells.

In various embodiments of the present invention the gases are selectedfrom a group consisting of oxygen (O₂), carbon dioxide (CO₂), nitrogen(N₂) and any combination thereof.

In a preferred embodiment of the present invention the ratio:percentageof each gas that need to be maintained is of O₂ from about 21% to about95%, CO₂ from about 0% to about 10% and N₂ from about 0% to about 80%.

In a preferred embodiment of the present invention the ratio:percentageof each gas that need to be maintained is O₂ at about 80%, CO₂ at about5% and N₂ at about 15%.

Bubble Trap

Unwanted bubbles inadvertently introduced into a microfluidic system cannegatively affect device operation and experimental outcome. This isespecially true for microfluidic perfusion culture systems, whichtypically require sterilization and pre-conditioning of the surfaceprior to cell seeding, time to allow for cell attachment, and then takeseveral days to observe the growth rate and cell morphologies. Bubblescan form at the connection between the device and tubing or can beintroduced when unplugging connections to transfer the device betweenthe microscope and incubator. The bubbles are cytotoxic to the cells andcan potentially rupture their cell membranes.

One solution to mitigate bubble-based device failure is to integratemicrofluidic features to prevent bubbles from entering critical areas ofa device. There are, in general, two different approaches: trappingversus debubbling. A bubble trap is a structure integrated into the flowsystem that halts further progress of a bubble through a device. It hasbeen demonstrated a simple, easily implemented bubble trap by making achamber at the connection point between external tubing and theirdevice. This approach has the advantage that device operation ismaintained while the bubbles are trapped. The alternative is to activelyremove the bubbles from the system. This is advantageous since thebubble trap does not remove bubbles from the system, so that when thebubble trap completely fills with bubbles, any additional bubbles wouldbe sent through the system. Active bubble removal can be achieved basedon gas permeability of the material forming the fluidic circuit 102(e.g., PDMS). In these embodiments, positive pressure is applied toforce bubbles out of fluidic circuit 102.

In an embodiment of the present invention, the system comprises adedicated part for the removal of bubbles selected from the groupconsisting of: bubble trap, debubbler, and any combination thereof.

Heat Exchanger

A heat exchanger is a device used to transfer heat between one or morefluids. The fluids may be separated by a solid wall, such as plastic ormetal tubing, to prevent mixing or they may be in direct contact. Theyare widely used in space heating, refrigeration, air conditioning, powerstations, chemical plants, petrochemical plants, petroleum refineries,natural-gas processing, and sewage treatment. The classic example of aheat exchanger is found in an internal combustion engine in which acirculating fluid known as engine coolant flows through radiator coilsand air flows past the coils, which cools the coolant and heats theincoming air(www.en.dot.wikipedia.dot.org/wiki/Heat_exchanger#Fluid_heat_exchangers—incorporatedherein as reference).

Flow arrangement: There are three primary classifications of heatexchangers according to their flow arrangement. In parallel-flow heatexchangers, the two fluids enter the exchanger at the same end, andtravel in parallel to one another to the other side. In counter-flowheat exchangers the fluids enter the exchanger from opposite ends. Thecounter current design is the most efficient, in that it can transferthe most heat from the heat (transfer) medium per unit mass due to thefact that the average temperature difference along any unit length ishigher. See countercurrent exchange. In a cross-flow heat exchanger, thefluids travel roughly perpendicular to one another through theexchanger.

For efficiency, heat exchangers are designed to maximize the surfacearea of the wall between the two fluids, while minimizing resistance tofluid flow through the exchanger. The exchanger's performance can alsobe affected by the addition of fins or corrugations in one or bothdirections, which increase surface area and may channel fluid flow orinduce turbulence.

The driving temperature across the heat transfer surface varies withposition, but an appropriate mean temperature can be defined. In mostsimple systems this is the “log mean temperature difference” (LMTD).Sometimes direct knowledge of the LMTD is not available and the Numberof Transfer Units (NTU) method is used.

Types: Double pipe heat exchangers are the simplest exchangers used inindustries. On one hand, these heat exchangers are cheap for both designand maintenance, making them a good choice for small industries. On theother hand, their low efficiency coupled with the high space occupied inlarge scales, has led modern industries to use more efficient heatexchangers like shell and tube or plate. However, since double pipe heatexchangers are simple, they are used to teach heat exchanger designbasics to students as the fundamental rules for all heat exchangers arethe same. To start the design of a double pipe heat exchanger, the firststep is to calculate the heat duty of the heat exchanger. It must benoted that for easier design, it's better to ignore heat loss to theenvironment for initial design.

Shell and tube heat exchanger: shell and tube heat exchangers consist ofseries of tubes. One set of these tubes contains the fluid that must beeither heated or cooled. The second fluid runs over the tubes that arebeing heated or cooled so that it can either provide the heat or absorbthe heat required. A set of tubes is called the tube bundle and can bemade up of several types of tubes: plain, longitudinally finned, etc.Shell and tube heat exchangers are typically used for high-pressureapplications (with pressures greater than 30 bar and temperaturesgreater than 260° C.). This is because the shell and tube heatexchangers are robust due to their shape.

Several thermal design features must be considered when designing thetubes in the shell and tube heat exchangers: There can be manyvariations on the shell and tube design. Typically, the ends of eachtube are connected to plenums (sometimes called water boxes) throughholes in tubesheets. The tubes may be straight or bent in the shape of aU, called U-tubes.

Tube diameter: Using a small tube diameter makes the heat exchanger botheconomical and compact. However, it is more likely for the heatexchanger to foul up faster and the small size makes mechanical cleaningof the fouling difficult. To prevail over the fouling and cleaningproblems, larger tube diameters can be used. Thus to determine the tubediameter, the available space, cost and fouling nature of the fluidsmust be considered.

Tube thickness: The thickness of the wall of the tubes is usuallydetermined to ensure:

There is enough room for corrosion

That flow-induced vibration has resistance

Axial strength

Availability of spare parts

Hoop strength (to withstand internal tube pressure)

Buckling strength (to withstand overpressure in the shell)

Tube length: heat exchangers are usually cheaper when they have asmaller shell diameter and a long tube length. Thus, typically there isan aim to make the heat exchanger as long as physically possible whilstnot exceeding production capabilities. However, there are manylimitations for this, including space available at the installation siteand the need to ensure tubes are available in lengths that are twice therequired length (so they can be withdrawn and replaced). Also, long,thin tubes are difficult to take out and replace.

Tube pitch: when designing the tubes, it is practical to ensure that thetube pitch (i.e., the centre-centre distance of adjoining tubes) is notless than 1.25 times the tubes' outside diameter. A larger tube pitchleads to a larger overall shell diameter, which leads to a moreexpensive heat exchanger.

Tube corrugation: this type of tubes, mainly used for the inner tubes,increases the turbulence of the fluids and the effect is very importantin the heat transfer giving a better performance.

Tube Layout: refers to how tubes are positioned within the shell. Thereare four main types of tube layout, which are, triangular (30°), rotatedtriangular (60°), square (90°) and rotated square (45°). The triangularpatterns are employed to give greater heat transfer as they force thefluid to flow in a more turbulent fashion around the piping. Squarepatterns are employed where high fouling is experienced and cleaning ismore regular.

Baffle Design: baffles are used in shell and tube heat exchangers todirect fluid across the tube bundle. They run perpendicularly to theshell and hold the bundle, preventing the tubes from sagging over a longlength. They can also prevent the tubes from vibrating. The most commontype of baffle is the segmental baffle. The semicircular segmentalbaffles are oriented at 180 degrees to the adjacent baffles forcing thefluid to flow upward and downwards between the tube bundle. Bafflespacing is of large thermodynamic concern when designing shell and tubeheat exchangers. Baffles must be spaced with consideration for theconversion of pressure drop and heat transfer. For thermo economicoptimization it is suggested that the baffles be spaced no closer than20% of the shell's inner diameter. Having baffles spaced too closelycauses a greater pressure drop because of flow redirection.Consequently, having the baffles spaced too far apart means that theremay be cooler spots in the corners between baffles. It is also importantto ensure the baffles are spaced close enough that the tubes do not sag.The other main type of baffle is the disc and doughnut baffle, whichconsists of two concentric baffles. An outer, wider baffle looks like adoughnut, whilst the inner baffle is shaped like a disk. This type ofbaffle forces the fluid to pass around each side of the disk thenthrough the doughnut baffle generating a different type of fluid flow.Fixed tube liquid-cooled heat exchangers especially suitable for marineand harsh applications can be assembled with brass shells, copper tubes,brass baffles, and forged brass integral end hubs.

Plate heat exchangers: another type of heat exchanger is the plate heatexchanger. These exchangers are composed of many thin, slightlyseparated plates that have very large surface areas and small fluid flowpassages for heat transfer. Advances in gasket and brazing technologyhave made the plate-type heat exchanger increasingly practical. In HVACapplications, large heat exchangers of this type are calledplate-and-frame; when used in open loops, these heat exchangers arenormally of the gasket type to allow periodic disassembly, cleaning, andinspection. There are many types of permanently bonded plate heatexchangers, such as dip-brazed, vacuum-brazed, and welded platevarieties, and they are often specified for closed-loop applicationssuch as refrigeration. Plate heat exchangers also differ in the types ofplates that are used, and in the configurations of those plates. Someplates may be stamped with “chevron”, dimpled, or other patterns, whereothers may have machined fins and/or grooves.

When compared to shell and tube exchangers, the stacked-platearrangement typically has lower volume and cost. Another differencebetween the two is that plate exchangers typically serve low to mediumpressure fluids, compared to medium and high pressures of shell andtube. A third and important difference is that plate exchangers employmore countercurrent flow rather than cross current flow, which allowslower approach temperature differences, high temperature changes, andincreased efficiencies.

Plate and shell heat exchanger: A third type of heat exchanger is aplate and shell heat exchanger, which combines plate heat exchanger withshell and tube heat exchanger technologies. The heart of the heatexchanger contains a fully welded circular plate pack made by pressingand cutting round plates and welding them together. Nozzles carry flowin and out of the platepack (the ‘Plate side’ flowpath). The fullywelded platepack is assembled into an outer shell that creates a secondflowpath (the ‘Shell side’). Plate and shell technology offers high heattransfer, high pressure, high operating temperature, uling and closeapproach temperature. In particular, it does completely without gaskets,which provides security against leakage at high pressures andtemperatures.

Adiabatic wheel heat exchanger: a fourth type of heat exchanger uses anintermediate fluid or solid store to hold heat, which is then moved tothe other side of the heat exchanger to be released. Two examples ofthis are adiabatic wheels, which consist of a large wheel with finethreads rotating through the hot and cold fluids, and fluid heatexchangers. Plate fin heat exchanger: this type of heat exchanger uses“sandwiched” passages containing fins to increase the effectiveness ofthe unit. The designs include crossflow and counterflow coupled withvarious fin configurations such as straight fins, offset fins and wavyfins.

Plate and fin heat exchangers are usually made of aluminum alloys, whichprovide high heat transfer efficiency. The material enables the systemto operate at a lower temperature difference and reduce the weight ofthe equipment. Plate and fin heat exchangers are mostly used for lowtemperature services such as natural gas, helium and oxygen liquefactionplants, air separation plants and transport industries such as motor andaircraft engines.

Advantages of Plate and Fin Heat Exchangers:

High heat transfer efficiency especially in gas treatment

Larger heat transfer area

Approximately 5 times lighter in weight than that of shell and tube heatexchanger.

Able to withstand high pressure

Disadvantages of Plate and Fin Heat Exchangers:

Might cause clogging as the pathways are very narrow

Difficult to clean the pathways

Aluminum alloys are susceptible to Mercury Liquid Embrittlement Failure

Pillow plate heat exchanger: a pillow plate exchanger is commonly usedin the dairy industry for cooling milk in large direct-expansionstainless steel bulk tanks. The pillow plate allows for cooling acrossnearly the entire surface area of the tank, without gaps that wouldoccur between pipes welded to the exterior of the tank.

The pillow plate is constructed using a thin sheet of metal spot-weldedto the surface of another thicker sheet of metal. The thin plate iswelded in a regular pattern of dots or with a serpentine pattern of weldlines. After welding the enclosed space is pressurized with sufficientforce to cause the thin metal to bulge out around the welds, providing aspace for heat exchanger liquids to flow, and creating a characteristicappearance of a swelled pillow formed out of metal.

Fluid heat exchangers: this is a heat exchanger with a gas passingupwards through a shower of fluid (often water), and the fluid is thentaken elsewhere before being cooled. This is commonly used for coolinggases whilst also removing certain impurities, thus solving two problemsat once. It is widely used in espresso machines as an energy-savingmethod of cooling super-heated water to use in the extraction ofespresso.

Waste heat recovery units: a Waste Heat Recovery Unit (WHRU) is a heatexchanger that recovers heat from a hot gas stream while transferring itto a working medium, typically water or oils. The hot gas stream can bethe exhaust gas from a gas turbine or a diesel engine or a waste gasfrom industry or refinery.

Big systems with high volume and temperature gas streams, typical inindustry, can benefit from Steam Rankine Cycle (SRC) in a WHRU, butthese cycles are too expensive for small systems. The recovery of heatfrom low temperature systems requires different working fluids thansteam.

An Organic Rankine Cycle (ORC) WHRU can be more efficient at lowtemperature range using Refrigerant that boil at lower temperatures thanwater. Typical organic refrigerants are Ammonia, Pentafluoropropane(R-245fa and R-245ca), and Toluene.

The refrigerant is boiled by the heat source in the Evaporator toproduce super-heated vapor. This fluid is expanded in the turbine toconvert thermal energy to kinetic energy, which is converted toelectricity in the electrical generator. This energy transfer processdecreases the temperature of the refrigerant that, in turn, condenses.The cycle is closed and completed using a pump to send the fluid back tothe evaporator.

Dynamic scraped surface heat exchanger: another type of heat exchangeris called “(dynamic) scraped surface heat exchanger”. This is mainlyused for heating or cooling with high-viscosity products,crystallization processes, evaporation and high-fouling applications.Long running times are achieved due to the continuous scraping of thesurface, thus avoiding fouling and achieving a sustainable heat transferrate during the process.

Phase-change heat exchangers: In addition to heating up or cooling downfluids in just a single phase, heat exchangers can be used either toheat a liquid to evaporate (or boil) it or used as condensers to cool avapor and condense it to a liquid. In chemical plants and refineries,reboilers used to heat incoming feed for distillation towers are oftenheat exchangers.

Distillation set-ups typically use condensers to condense distillatevapors back into liquid.

Power plants that use steam-driven turbines commonly use heat exchangersto boil water into steam. Heat exchangers or similar units for producingsteam from water are often called boilers or steam generators.

In the nuclear power plants called pressurized water reactors, speciallarge heat exchangers pass heat from the primary (reactor plant) systemto the secondary (steam plant) system, producing steam from water in theprocess. These are called steam generators. All fossil-fueled andnuclear power plants using steam-driven turbines have surface condensersto convert the exhaust steam from the turbines into condensate (water)for re-use.

To conserve energy and cooling capacity in chemical and other plants,regenerative heat exchangers can transfer heat from a stream that mustbe cooled to another stream that must be heated, such as distillatecooling and reboiler feed pre-heating.

This term can also refer to heat exchangers that contain a materialwithin their structure that has a change of phase. This is usually asolid to liquid phase due to the small volume difference between thesestates. This change of phase effectively acts as a buffer because itoccurs at a constant temperature but still allows for the heat exchangerto accept additional heat. One example where this has been investigatedis for use in high power aircraft electronics.

Heat exchangers functioning in multiphase flow regimes may be subject tothe Ledinegg instability.

Direct contact heat exchangers: Direct contact heat exchangers involveheat transfer between hot and cold streams of two phases in the absenceof a separating wall. Thus such heat exchangers can be classified as:

Gas—liquid

Immiscible liquid—liquid

Solid-liquid or solid—gas

Most direct contact heat exchangers fall under the Gas—Liquid category,where heat is transferred between a gas and liquid in the form of drops,films or sprays.

Such types of heat exchangers are used predominantly in airconditioning, humidification, industrial hot water heating, watercooling and condensing plants.

TABLE 1 Continuous Driving Change of Phases phase force phase ExamplesGas- Gas Gravity No Spray columns, packed Liquid columns Yes Coolingtowers, falling droplet evaporators Forced No Spray coolers/quenchersLiquid Yes Spray condensers/ flow evaporation, jet condensers LiquidGravity No Bubble columns, perforated tray columns Yes Bubble columncondensers Forced No Gas spargers Gas flow Yes Direct contactevaporators, submerged combustion

Microchannel heat exchangers: Micro heat exchangers, Micro-scale heatexchangers, or microstructured heat exchangers are heat exchangers inwhich (at least one) fluid flows in lateral confinements with typicaldimensions below 1 mm. The most typical such confinement aremicrochannels, which are channels with a hydraulic diameter below 1 mm.Microchannel heat exchangers can be made from metal, ceramic, and evenlow-cost plastic. Microchannel heat exchangers can be used for manyapplications including:

high-performance aircraft gas turbine engines

heat pumps

air conditioning

heat recovery ventilators

Helical-coil heat exchangers: Although double-pipe heat exchangers arethe simplest to design, the better choice in the following cases wouldbe the helical-coil heat exchanger (HCHE):

The main advantage of the HCHE, like that for the SHE, is its highlyefficient use of space, especially when it's limited and not enoughstraight pipe can be laid.

Under conditions of low flowrates (or laminar flow), such that that thetypical shell-and-tube exchangers have low heat-transfer coefficientsand becoming uneconomical.

When there is low pressure in one of the fluids, usually fromaccumulated pressure drops in other process equipment.

When one of the fluids has components in multiple phases (solids,liquids, and gases), which tends to create mechanical problems duringoperations, such as plugging of small-diameter tubes. Cleaning ofhelical coils for these multiple-phase fluids can prove to be moredifficult than its shell and tube counterpart; however the helical coilunit would require cleaning less often.

These have been used in the nuclear industry as a method for exchangingheat in a sodium system for large liquid metal fast breeder reactorssince the early 1970s, using an HCHE device invented by Charles E.Boardman and John H. Germer. There are several simple methods fordesigning HCHE for all types of manufacturing industries, such as usingthe Ramachandra K. Patil (et al.) method from India and the Scott S.Haraburda method from the United States.

However, these are based upon assumptions of estimating inside heattransfer coefficient, predicting flow around the outside of the coil,and upon constant heat flux. Yet, recent experimental data revealed thatthe empirical correlations are quite in agreement for designing circularand square pattern HCHEs. During studies published in 2015, severalresearchers found that the boundary conditions of the outer wall ofexchangers were essentially constant heat flux conditions in power plantboilers, condensers and evaporators; while convective heat transferconditions were more appropriate in food, automobile and processindustries.

In an embodiment of the present invention, the system comprises adedicated part for the heat conservancy of the medium in a form of aheat exchanger selected from the group consisting of: shell and tubeheat exchanger, plate heat exchanger, plate and shell heat exchanger,adiabatic wheel heat exchanger, plate fin heat exchanger, pillow plateheat exchanger, fluid heat exchanger, waste heat recovery units, dynamicscraped surface heat exchanger, phase-change heat exchanger, directcontact heat exchanger, microchannel heat exchanger, helical-coil heatexchanger, spiral heat exchanger, and any combination thereof.

Dialyzer

A dialyzer is a machine equipped with a semipermeable membrane and usedfor performing dialysis. Dialysis is the process of diffusion of solutesthrough a semipermeable membrane from a liquid with higher soluteconcentration on one side of the membrane to a liquid with a lowerconcentration on the other side. The membranes are semipermeable becausethey allow some molecules to pass while preventing others from passing.The process has long been used for the molecular separation of smallmolecules from macromolecules(www.dot.spectrumlabs.dot.com/lit/abc.dot.pdf, incorporated hereinafteras reference) and for extracorporeal support (kidney dialysis,www.en.dot.wikipedia.dot.org/wiki/Dialysis, incorporated hereinafter asreference).

Common dialysis applications utilize tubular forms of membranes andinvolve placing a “sample” inside the membrane and a “buffer” outsidethe membrane. The process is run until the desired degree of separationis attained. Molecules smaller than the pores will eventually be equallydistributed between the two solutions. Usually, a very large volume ofbuffer is chosen so that the permeable species are greatly diluted andtherefore reduced-to very small concentrations in the remaining samplesolution. Commonly, dialysis processes require several hours tocomplete.

Dialysis Membranes: advances in dialysis membrane development were madeas a result of research to provide relief from renal disease by means ofhemodialysis, a pressure driven rather than concentration gradientdriven process. Greater membrane permeability was achieved through theuse of cellulose ester. These solutions could be formulated to yield awider range of pore sizes. Cellulose ester membranes are now widely usedfor clinical and laboratory dialysis. Membranes used for dialysis havepore sizes ranging from 100 to 300,000 Daltons (1 to 300 kDa). Samplevolumes have also been greatly reduced to allow dialysis of smallquantities of precious samples, particularly where maintaining enzymeactivity is desired.

Factors that Affect the Rate of Dialysis

Molecular Weight Cut Off and Selectivity: dialysis membranes arecharacterized by molecular weight cut off (MWCO). MWCO is determined bytesting the degree of permeability for several solutes of differentmolecular weights. The MWCO rating for the membrane is the molecularweight of the smallest solute that is 90% retained in a 17-hour dialysistest. Molecular weight cut off ratings are used as a guide and not anabsolute prediction of performance with every type of solute. A membraneMWCO size rating should be chosen as high as possible in order toachieve the maximum dialysis rate while still preventing the loss of thedesired solute. Plotting the results of a MWCO test in the form ofretention versus the solute molecular weight would ideally produce asigmoid curve. The steepness of the curve is a measure of theselectivity of the membrane.

Flux and Permeation Rate: the driving force for laboratory dialysis isthe concentration difference across the membrane. The flux (orpermeation rate) is directly proportional to the concentrationdifference, i.e. the greater the difference, the greater the rate.However, the dialysis rate is also influenced by other variables suchas:

Diffusion coefficient: different size molecules pass through a membraneat different rates. Larger molecules have a smaller coefficient and alower rate of diffusion across the membrane.

Molecular shape and charge: linear molecules permeate faster thanglobular molecules. The pH and ionic strength also affect the rate ofdialysis.

Concentration polarization: As molecules diffuse across a membrane, theyfirst move through the bulk of the sample solution to the surface of themembrane. The thin region next to the membrane has a higherconcentration of solutes than the bulk solution. This build up is termed“concentration polarization” and is caused by depletion of smallmolecules at the surface of the membrane. This polarized layer causesresistance to the movement of molecules across the membrane. Finally,after passing through the membrane, the molecule often meets a thinlayer of concentration higher than the bulk solution, further slowingthe passage. These layers which form on either side of the membrane arecalled “fluid boundary layers” or “gel layers”.

Flow direction and agitation of the solution: sample and buffer thatflow perpendicular (or normal) might cause the membrane to plug. Sampleand buffer mixing during dialysis can reduce this phenomenon. Mixing canbe achieved by either stirring or by passing the sample parallel (ortangential) to the membrane. Parallel flow promotes higher permeationrates. The higher the stirring rate, the higher the dialysis rates(Concentration polarization is reduced by increased stirring rates).

Temperature: higher temperatures promote more rapid molecular movementand therefore increase diffusion rate.

Membrane thickness: membrane properties effect the dialysis rate.Thicker membranes will require a longer time for molecules to passthrough.

Membrane surface area: the larger the membrane area, the faster thedialysis rate.

Hydrodynamic properties: viscosity of the fluid and the membraneporosity affect the permeation rate. Low viscosity and high porosity areideal for higher rates.

MWCO Selection: selecting of the correct molecular weight cut off (MWCO)of the membrane is based on the size of the molecular weight of themacromolecules to be retained inside the membrane and the molecularweight of the molecules to be removed. The ratio of the two molecularweights should be a minimum 25 to 1 to achieve the maximum 90%retention.

Tubular Membrane “flat width” Selection: smaller tubing will dialyzemore quickly than larger tubing. The latter will dialyze more slowly dueto the longer diffusion distances involved.

Albumin is the main carrier protein of growth factors, hormones andfatty acids, and a major cost driver of liquid medium. The system of thepresent invention is optionally and preferably designed to retainalbumin (MW about 66.4 kDa), achievable with a target MWCO of 30 kDa.

In an embodiment of the present invention, the system comprises adialyzer with surface ranging from 15 to 20,000 cm² membrane area and amolecular weight cutoff ranging from 10 to 60 kDa.

Dialysate

Dialysate or diffusate is the fluid and solutes in a dialysis processthat passes through the membrane in dialysis.

In an embodiment of the present invention, the system comprises adialysate containing glucose, insulin and growth factors in serum-freemedium. Depending on the type of cells being grown in the chamber, adifferent content of dialysate is prepared in order to respond to thespecific needs of the growing cells.

Filtering

Filtering can be effected according to some embodiments of the presentinvention by any type of filter that can remove contaminants andimpurities. Representative examples including, without limitation,carbon filtering and zeolite filtering.

Carbon filtering is a method of filtering that uses a bed of activatedcarbon to remove contaminants and impurities, using chemical adsorption(www.en.dot.wikipedia.dot.org/wiki/Carbon_filtering, incorporatedhereinafter as reference).

Each particle/granule of carbon provides a large surface area/porestructure, allowing contaminants the maximum possible exposure to theactive sites within the filter media. One pound (454 g) of activatedcarbon contains a surface area of approximately 100 acres (40 Hectares).

Activated carbon works via a process called adsorption, wherebypollutant molecules in the fluid to be treated are trapped inside thepore structure of the carbon substrate. Carbon filtering is commonlyused for water purification, in air purifiers and industrial gasprocessing, for example the removal of siloxanes and hydrogen sulfidefrom biogas. It is also used in a number of other applications,including respirator masks, the purification of sugarcane and in therecovery of precious metals, especially gold. Active charcoal carbonfilters are most effective at removing chlorine, sediment, volatileorganic compounds (VOCs), taste and odor from water. They are noteffective at removing minerals, salts, and dissolved inorganiccompounds.

Typical particle sizes that can be removed by carbon filters range from0.5 to 50 micrometres. The particle size will be used as part of thefilter description. The efficacy of a carbon filter is also based uponthe flow rate regulation. When the water is allowed to flow through thefilter at a slower rate, the contaminants are exposed to the filtermedia for a longer amount of time.

There are 2 predominant types of carbon filters used in the filtrationindustry: powdered block filters and granular activated filters. Ingeneral, carbon block filters are more effective at removing a largernumber of contaminants, based upon the increased surface area of carbon.Many carbon filters also use secondary media such as silver to preventbacteria growth within the filter. Alternatively, the activated carbonitself may be impregnated with silver to provide this bacteriostaticproperty.

Factors that affect the performance of activated carbon are(www.dot.watertreatmentguide.dot.com/activated_carbon_filtration.dot.htm,incorporated hereinafter as reference):

Molecular weight: as the molecular weight increases, the activatedcarbon adsorbs more effectively because the molecules are lea soluble inwater. However, the pore structure of the carbon must be large enough toallow the molecules to migrate within. A mixture of high and lowmolecular weight molecules should be designed for the removal of themore difficult species.

pH: most organics are less soluble and more readily adsorbed at a lowerpH. As the pH increases, removal decreases. A rule of thumb is toincrease the size of the carbon bed by twenty percent for every pH unitabove neutral (7.0).

Contaminant concentration: the higher the contaminant concentration, thegreater the removal capacity of activated carbon. The contaminantmolecule is more likely to diffuse into a pore and become adsorbed. Asconcentrations increase, however, so do effluent leakages. The upperlimit for contaminants is a few hundred parts per million. Highercontaminant concentration may require more contact time with theactivated carbon. Also, the removal of organics is enhanced by thepresence of hardness in the water, so whenever possible, place activatedcarbon units upstream of the ion removal units. This is usually the caseanyway since activated carbon is often used upstream of ion exchange ormembranes to remove chlorine.

Particle size: activated carbon is commonly available in 8 by 30 mesh(largest), 12 by 40 mesh (most common), and 20 by 50 mesh (finest). Thefiner mesh gives the best contact and better removal, but at the expenseof higher pressure drop. A rule of thumb here is that the 8 by 30 meshgives two to three times better removal than the 12 by 40, and 10 to 20times better kinetic removal than the 8 by 30 mesh.

Flow rate: generally, the lower the flow rate, the more time thecontaminant will have to diffuse into a pore and be adsorbed. Adsorptionby activated carbon is almost always improved by a longer contact time.Again, in general terms, a carbon bed of 20 by 50 mesh can be run attwice the flow rate of a bed of 12 by 40 mesh, and a carbon bed of 12 by40 mesh can be run at twice the flow rate of a bed of 8 by 30 mesh.

Temperature: higher water temperatures decrease the solution viscosityand can increase die diffusion rate, thereby increasing adsorption.Higher temperatures can also disrupt the adsorptive bond and slightlydecrease adsorption. It depends on the organic compound being removed,but generally, lower temperatures seem to favor adsorption. In anembodiment of the present invention, the system comprises a carbonfilter adapted to clean toxins from present in the dialysate.

When zeolite filtering is employed, the portion of the perfusionsolution that enters system 200 is passed through zeolite to absorb theammonia in the solution. Preferably, granular zeolite is employed. Theterm zeolite is intended to encompass hydrated aluminosilicate mineralsthat have a micro-porous structure. Natural zeolites are formed wherevolcanic rocks and ash layers react with alkaline ground water. Granularzeolites suitable for use in the present invention can, for example, besourced from Zeolite Australia Pty Ltd (PO Box 6 Werris Creek NSW 2341,Australia).

Sensors

In a preferred embodiment of the present invention, system 1000comprises one or more active sensors (not shown in FIGS. 1A-B, see FIG.9) that allow continuous monitoring of the cells growing therein. Someexamples of sensors comprise, but are not limited to, temperaturesensors, pH sensor, volume sensor, video apparatuses, flow sensor,optical sensors, weight sensor, glucose sensor, and protein contentsensor.

In a preferred embodiment of the present invention, the system isconnected to a main computer, having a non-transitory computer readablemedium (CRM), that operates automatically all the daily necessities ofthe system and provides real-time alarms to dedicated operators. Themain computer can be connected and operated remotely via internet/cloudservices. In a second embodiment the system is self-contained, with datafrom sensors analyzed by a local central processing unit (CPU), whichchanges input parameters such a nutrient, flow, pressure or temperatureto adjust cell growth and sensor signal to within desired parameter set,maintaining growth homeostasis.

In a preferred embodiment of the present invention, the system comprisesa Closed-loop perfusion circuit composed of a primary perfusion circuitand a secondary dialysis circuit for nutrient and toxin exchange. Theprimary circuit includes culture medium perfusate that is recirculatedusing a peristaltic pump through a jacketed cell growth chamber, amembrane oxygenator, a heat exchanger, and a bubble trap. The oxygenatoris gassed with a mixture of 80% O₂/5% CO₂/15% N₂ maintaining constantpH. A fraction of the perfusate is diverted to secondary circuit througha dialyzer with a 2200 cm² membrane area and a 30 kDa molecular weightcutoff at a rate of 3 mL/min/gram cells. The secondary circuit dialyzedthe perfusate by counter-current exposure to protein-free dialysate,recirculated through a carbon filter using a third peristaltic pump.Temperature within the system is maintained at 37° C. All the system

Cell Types

Several types of cells can be grown in the closed-loop perfusion circuitdisclosed in the present invention.

Primary Cell Source

Chicken embryonic fibroblasts are widely used for the production ofviruses and vaccines. Together with chicken embryonic liver cells theyare produced from specific pathogen-free (SPF) embryos and sold byCharles River Laboratories (Wilmington, Mass.) and other companies.While chicken liver cells show limited proliferation in culture, liketheir mammalian counterparts, chicken fibroblasts can undergo over 30population doublings, producing about 2.6 ton of cells beforespontaneously immortalizing without becoming tumorigenic. Spontaneouslytransformed chicken fibroblasts, such as the CSIF cell line generated bythe present inventor (e.g., as described in Example 5 of the Examplessection which follows), UMNSAH/DF-1 (CRL-12203) can be bought directlyfrom ATTC (Manassas, Va.). While the growth potential of fibroblast isexcellent, the cells primarily form inedible connective tissue.

Chicken embryonic endothelium can be easily isolated but their growthpotential is unknown and can be organ specific. Mouse micro-vascularcells can undergo 30 population doublings, while human cells seldom pass12 population doublings. Chicken embryonic muscle cells (myocytes) canbe similar isolated but have a very limited growth potential. Mouse andhuman cells seldom pass 12 population doublings. Myogenesis, theformation of new muscle tissue, is uncommon past the neonatal stage oflife in most species. Small molecules can conceptually be used tomodulate this behavior.

Pluripotent Stem Cell Source

Numerous groups produced chicken embryonic stem cells (cESC) over thelast decade (3). Cells are isolated from fertilized chicken eggs and areessentially immortal. Chicken induced pluripotent stem cells (ciPSC)were produced from quail embryonic fibroblasts by reprogramming factorsOCT4, NANOG, SOX2, LIN28, KLF4, and C-MYC (4) and more recently chickenfibroblasts using OCT4, KLF4, and C-MYC (5). Cells are essentiallyimmortal but are genetically engineered.

Recently, mouse pluripotent stem cells were induced from fibroblastsusing small molecules (6) permitting the differentiation of multiplecell types, including myocytes, hepatocytes, and endothelial cells aswell as complex embryoid bodies. Chemical induction of ciPSC offers analternative approach to convert fibroblasts to other cell types.

Small Molecule-Based Reprogramming

Chemical compounds offer an attractive alternative to growth factors andgenetic engineering that are generally used to support cell growth, orto switch one cell type to another through reprogramming ordifferentiation. Small molecules are less expensive, have lowerlot-to-lot variability, are non-immunogenic and are much more stable. Inone study, Shan and colleagues used a high content screen to identifyFPH1 and FPH2, small molecules that promoted proliferation of primaryhuman hepatocytes (7). This approach is appealing, as small moleculescould replace growth factors serum-free medium formulations,dramatically reducing costs while increasing safety.

In a more recent study, Cao and colleagues identified a combination of 9compounds that induced human fibroblasts to turn into cardiomyocytes(8), while others used a 7 compound combination to transform mouse cells(9). Considering many of the signaling pathways are conserved, arelatively similar combination could be used to transform chickenfibroblasts into myocytes.

Animal Product Free Culture Medium

As mentioned above, cell culture medium often contains fetal bovineserum (FBS) that provides attachment factors, fatty acids, growthfactors, hormones, and albumin. FBS can usually be replaced with serumreplacement (e.g. KO-serum) that is composed of amino acids, vitamins,and trace elements in addition to transferrin, insulin, and lipid-richbovine serum albumin. While both transferrin and insulin are produced inbacteria using recombinant technology, albumin is usually animalderived. However, plant and bacteria-derived recombinant human albumin(e.g. Cellastim™) are available through several companies, includingSigma-Aldrich (St. Louis, Mo.).

Chicken fibroblast medium is traditionally composed of M199 mediumsupplemented with 10% FBS, tryptose phosphate and glutamine. However,serum-free medium for the growth of mammalian fibroblasts is now readilyavailable. Medium is composed of M199 supplemented with 0.5 mg/mLalbumin, 0.6 μM linoleic acid, 0.6 μg/mL lecithin, 5 ng/mL bFGF, 5 ng/mLEGF, 30 pg/mL TGF131, 7.5 mM glutamine, 1 μg/mL hydrocortisone, 50 μg/mLascorbic acid, and 5 μg/mL insulin. This medium PCS-201-040 is availablefrom ATCC (Manassas, Va.) and is reported to support 4-fold fasterproliferation of human fibroblasts. Chicken hepatocytes are similarlysupported by a serum-free culture medium designed for human and mousehepatocytes. Medium is composed of Williams E basal medium supplementedwith albumin, insulin, transferrin, and hydrocortisone.

Perfused culture medium can also include an oxygen carrier. Hemoglobinbased oxygen carriers(www.en.dot.wikipedia.dot.org/wiki/Haemoglobin-based_oxygen_carriers,incorporated hereinafter as reference) include hemoglobin derivativeseither recombinant or chemically modified, encapsulated hemoglobin ormodified (e.g. cross-linked) red blood cells. Alternatives includePerfluorocarbon based alternatives such as those developed in Nahmias etal. (11) (www.en.dot.wikipedia.dot.org/wiki/Blood_substitute#Currenttherapeutics, incorporated hereinafter as reference)

The present inventor has uncovered that a spontaneously immortalizedfibroblast, such as chicken fibroblast, can be used to generate fat andmuscle cells in-vitro for the generation of edible meat. In addition,the present inventor has uncovered that primary or spontaneouslyimmortalized endothelial cell can be co-cultured with the muscle and fatcells in order to form an edible meat with vascular-like network (tissuevessels) in which the endothelial cells serve as vessels for transfer ofnutrients and gasses, such as glucose and oxygen. Example 5 of theExamples section which follows demonstrates the isolation and generationof a spontaneously immortalized chicken embryonic fibroblast cell linehaving a doubling time of 18±2 hours and at least 90 populationdoublings (PDs) (FIG. 2E). In addition, as is further described inExample 6 of the Examples section which follows, the present inventorhas generated, following laborious experimentations, a serum-freeculture medium which can maintain the spontaneously immortalized chickenfibroblast cell line under conditions devoid of any animal and/or humancontaminants, while maintaining the fibroblasts in a proliferative statefor at least 90 population doublings (FIGS. 3B-F). The present inventorhas further envisaged that small molecules can substitute at least someof the components included in the serum-free medium (Examples 2, 3 and 6of the Examples section which follows). The present inventor was able tosuccessfully generate fully functional adipocyte cells, characterized bya compact (not elongated) shape and the accumulation of neutral lipidcontent from the spontaneously immortalized chicken embryonic fibroblastcell line in a defined serum-free culture medium which includes oleicacid and a small molecule which activates PPAR-gamma such as IBMX orRosiglitazone (FIGS. 4A-D, Example 7). The present inventor furthergenerated myocyte cells by upregulating the expression level andactivity of the MyoD1 and/or Myogenin polypeptides within thespontaneously immortalized chicken embryonic fibroblast cell line(Examples 3 and 8, FIGS. 6-8, 12 and 5A-E). In addition, as shown indescribed in Examples 3 and 8 of the Examples section which follows, thepresent inventor describes a screen for small molecules capable ofconverting the spontaneously immortalized chicken embryonic fibroblastcell line into myocytes using the rat myosin light chain-3promoter-enhancer reporter construct (rMLC3-GFP; FIG. 8). Furthermore,the present inventor shows that spontaneously immortalized endothelialcells (e.g., reaching at least 120 population doublings; Example 11),which were co-cultured in serum-free and antibiotic-free culture mediumwith the spontaneously immortalized fibroblast cell line (Example 12)formed vascular network formation and close cell-cell interactions(FIGS. 11A-C). Furthermore, the present inventor describes a hybridplant-based meat substitute product with in-vitro generated fat (Example9), and patty or nuggets from the cultured fibroblasts which wereinduced towards differentiation into muscle and/or fat cells in asuspension culture devoid of microcarriers (Example 10).

According to an aspect of some embodiments of the invention, there isprovided an in-vitro method of generating an adipocyte cell from afibroblast, comprising culturing a spontaneously immortalized fibroblastin a serum-free medium comprising oleic acid and a peroxisomeproliferator-activated receptor gamma (PPAR-gamma) agonist or activatorthereof, thereby generating the adipocyte cell.

As used herein the phrase “spontaneously immortalized fibroblast” refersto a fibroblast cell which is capable of undergoing unlimited celldivision, and preferably also cell expansion, without being subjected toman-induced mutation e.g., genetic manipulation, causing theimmortalization.

It should be noted that normally, primary fibroblast cells are capableof a limited cell division, and thus undergo cellular senescence afterabout 30 population doublings (e.g., 10 passages). Methods of generatingimmortalized fibroblastoid cell lines include genetic manipulation byintroduction of a telomerase gene, or SV40, or HPVE6/E7 gene using knownmethods.

According to some embodiments of the invention, the fibroblast is anavian fibroblast.

According to some embodiments of the invention, the avian is selectedfrom the group consisting of: chicken, duck, goose, and quail.

According to some embodiments of the invention, the fibroblast is achicken embryonic fibroblast.

According to some embodiments of the invention, the spontaneouslyimmortalized fibroblast is non-genetically modified.

As used herein the phrase “non-genetically modified” refers to not beingsubject to man-made genetic manipulation (e.g., transformation) of thecell.

PPAR is subfamily of the nuclear receptor superfamily of transcriptionfactors, plays important roles in lipid and glucose metabolism, and hasbeen implicated in obesity-related metabolic diseases such ashyperlipidemia, insulin resistance, and coronary artery disease.

PPARγ (peroxisome proliferator-activated receptor gamma) is a fattyacid-activated member of the PPAR subfamily. It is expressed at lowlevels in most physiological systems, including the central nervoussystem (CNS), endocrine system, gastrointestinal system, reproductivesystem, cardiopulmonary system and metabolic tissues, but is most highlyexpressed in brown and white adipose tissue (Elbrecht A, et al. 1996;“Molecular cloning, expression and characterization of human peroxisomeproliferator activated receptors gamma 1 and gamma 2”. Biochem. Biophys.Res. Commun. 224 431-7 V).

As used herein the phrase “PPAR-gamma activator” refers to an agentwhich induces the signaling pathway of PPAR-gamma leading to activationof PPAR-gamma.

According to some embodiments of the invention, an activator ofPPAR-gamma does not need to directly bind the ligand-binding domain ofPPAR-gamma, but can induce the PPAR-gamma signaling pathway leading toactivation of PPAR-gamma by endogenous ligand(s).

For example, a PPAR-gamma activator can be PPAR-gamma agonist.

As used herein the phrase “PPAR-gamma agonist” refers to an agent whichbinds to the ligand-binding domain of PPAR-gamma.

It should be noted that upon binding of the agonist to theligand-binding domain of PPAR-gamma, the PPAR-gamma protein undergoes aconformational change resulting in activation of PPAR-gamma.

For example, activation of PPAR-gamma (a transcription factor) can bedetected by monitoring expression of PPAR-gamma target genes.

Methods of qualifying agonists or activators of PPAR-gamma include, butare not limited to using a GAL4-PPAR-gamma reporter, a LanthaScreenTR-FRET competitive binding assay (ThermoFisher, PV4894), using aGFP-reporter driven by PPAR response element (PPRE), or by checking theexpression of target genes, essentially as described in Goldwasser etal. PLoS One 2010, Volume 5, Issue 8, e12399, which is fullyincorporated herein by reference).

Non-limiting examples of PPARγ (gamma) target genes, include genesrelated to adipogenesis (e.g., ADIPOQ, LPL, NR1H3, and UCP1); genesrelated to fatty Acid Metabolism (e.g., ACADL, ACADM, ACOX1, ACOX3,ACSL1, ACSL3, ACSL4, ACSL5, CPT1A, CPT1B, CPT2, CYP27A1, CYP4A11,CYP7A1, EHHADH, FADS2, GK, and SCD); genes related to lipid transport(e.g., ADIPOQ, ANGPTL4, APOE, DGAT1, LPL, NR1H3, and OLR1); genesrelated to cell proliferation (e.g., CLU, ELN, HSPD1, and TXNIP); genesrelated to insulin signaling (e.g., CPT1A, DGAT1, PCK1, and SORBS1) andother genes such as MMP9 and PCK1.

According to some embodiments of the invention, the PPAR-gamma agonistor activator is a small molecule.

According to some embodiments of the invention, the small molecule isselected from the group consisting of Thiazolidinedione,3-Isobutyl-1-methylxanthine (IBMX), phenamil, GW7845, RG14620, andHarmine.

Thiazolidinediones (also known as “Glitazones”) are a class ofmedications that act by activating PPARs (peroxisomeproliferator-activated receptors), with greatest specificity for PPARγ(PPAR-gamma, PPARG). The endogenous ligands for these receptors are freefatty acids (FFAs) and eicosanoids.

According to some embodiments of the invention, the Thiazolidinedione isprovided at a concentration in the range of about 20 nM to about 120 μM,e.g., from 50 nM to 100 μM, e.g., from 100 nM to 50 μM, e.g., from 1 μMto 50 μM, e.g., in the range of 0.5-30 μM, e.g., in the range of 0.5-25μM, e.g., about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about 15μM.

According to some embodiments of the invention, the Thiazolidinedione isselected from the group consisting of Pioglitazone (Actos),Rosiglitazone (Avandia), Lobeglitazone (Dulie), Troglitazone (Rezulin),Ciglitazone, Darglitazone, Englitazone, Netoglitazone, andRivoglitazone.

According to some embodiments of the invention, the small molecule isrosiglitazone.

According to some embodiments of the invention, the concentration ofrosiglitazone is between 1-10 μM, e.g., about 5 μM.

According to some embodiments of the invention, the concentration oftroglitazone is between 0.5-10 μM, e.g., about 0.5-5 μM, e.g., about 1μM.

According to some embodiments of the invention, the PPAR-gamma agonistor activator is selenium.

Oleic acid is a naturally-occurring fatty acid, classified asmonounsaturated omega-9 fatty acid, abbreviated with a lipid number of18:1 cis-9.

According to some embodiments of the invention, the concentration ofoleic acid which is used in the serum-free medium of some embodiments ofthe invention is from about 50 μM to about 1000 μM, e.g., between200-400 μM.

According to some embodiments of the invention, the culturing of thefibroblast is for at least 4 days, e.g., for at least 5, 6, 7, 8, 9, 10,15, 20 or more days.

It should be noted that for generation of a cultured edible meat themedium used in the method of generating an adipocyte cell should bewell-defined, and serum-free. Well-defined culture medium can beprepared by using recombinant, and/or synthetically and/or purifiedagents. Since serum is obtained from a living organism, e.g., a humanbeing or an animal, it is subject to batch-to-batch variations, and mayfurther include animal or human contaminants, such as bacterial, viralor fungal infections. Accordingly, it is preferred to use a serum-freemedium.

According to some embodiments of the invention, the serum-free medium isdevoid of animal contaminants.

According to some embodiments of the invention, the serum-free medium isdevoid of human contaminants.

According to some embodiments of the invention, the serum-free medium isdevoid any antibiotic drug.

According to some embodiments of the invention, for the adipocytedifferentiation the serum-free medium can include insulin, andoptionally also bFGF.

According to some embodiments of the invention, for the adipocytedifferentiation the serum-free medium can include selenium, andoptionally also insulin.

According to some embodiments of the invention, the serum-free mediumfor culturing the spontaneously immortalized chicken fibroblastscomprises insulin or a substitute thereof, and basic fibroblast growthfactor (bFGF) or a substitute thereof, and at least one additional agentselected from the group consisting of dexamethasone, transferrin,selenium, epidermal growth factor (EGF) or a substitute thereof, andProstaglandin E2 (PGE2).

As used herein the term “insulin” refers to the mature insulinpolypeptide having A chain and B chain, which are covalently linked viatwo disulfide bonds. Also known as CAS Number 11061-68-0; EC Number234-279-7; MDL number MFCD00131380. The precursor polypeptidepreproinsulin is cleaved to remove the precursor signal peptide, andthen the proinsulin is post-translationally cleaved into three peptides:the B chain and A chain peptides, which are covalently linked via twodisulfide bonds to form insulin, and C-peptide. Binding of insulin tothe insulin receptor (INSR) stimulates glucose uptake. There are 4polypeptide variants, encoding the same protein: variant 1 [GenBankAccession No. NM_000207.2 (SEQ ID NO: 13), GenBank Accession No.NP_000198.1 (SEQ ID NO: 14)], variant 2 [GenBank Accession No.NM_001185097.1 (SEQ ID NO: 15), GenBank Accession No. NP_001172026.1(SEQ ID NO: 16)]; variant 3 [GenBank Accession No. NM_001185098.1 (SEQID NO: 17), GenBank Accession No. NP_001172027.1 (SEQ ID NO: 18)]; andvariant 4 [GenBank Accession No. NM_001291897.1 (SEQ ID NO: 19), GenBankAccession No. NP_001278826.1 (SEQ ID NO: 20)]. Insulin can be providedfrom various suppliers such as Sigma-Aldrich (e.g., recombinant humaninsulin Catalogue Number 91077C).

According to some embodiments of the invention, the insulin substitutecomprises IGF-1 (Sigma 1146) or a stabilized Long R3 IGF-1 (Sigma I1271)According to some embodiments of the invention, the insulin is providedat a concentration of 2.5×10⁻⁵ IU/mL to 1 IU/mL, e.g., between 0.1 IU/mLto about 0.5 IU/mL, e.g., about 0.24-0.3 IU/mL. It should be noted thatIU/mL is an abbreviation of “International Units Per Millilitre(milliliter)”.

Dexamethasone is a corticosteroid medication which can be obtained fromvarious suppliers such as Ark Pharm, Inc., Sigma-Aldrich, Parchem, andAvaChem Scientific.

According to some embodiments of the invention, the dexamethasone isprovided at a concentration of about 0.01 nM to about 100 μM, e.g., fromabout 0.01 nM to about 10 μM, e.g., from 4 nM to about 10 μM, e.g.,between 70-120 nM, e.g., about 100 nM (0.1 μM).

According to some embodiments of the invention, the medium includesBasic fibroblast growth factor (bFGF) or a substitute thereof, such as asmall molecule or a synthetic agonist of the FGF-signaling pathway.

Basic fibroblast growth factor (also known as bFGF, FGF2 or FGF-β) is amember of the fibroblast growth factor family. BFGF [(e.g., human bFGFpolypeptide GenBank Accession No. NP_001997.5 (SEQ ID NO:21); human bFGFpolynucleotide GenBank Accession No. NM_002006.4 (SEQ ID NO: 22)] can beobtained from various commercial sources such as Cell Sciences®, Canton,Mass., USA (e.g., Catalogue numbers CRF001A and CRF001B), InvitrogenCorporation products, Grand Island N.Y., USA (e.g., Catalogue numbers:PHG0261, PHG0263, PHG0266 and PHG0264), ProSpec-Tany TechnoGene Ltd.Rehovot, Israel (e.g., Catalogue number: CYT-218), and Sigma, St Louis,Mo., USA (e.g., catalogue number: F0291).

According to some embodiments of the invention, the bFGF is provided ata concentration of 0.1-100 ng/ml, e.g., about 0.1-30 ng/ml, e.g., about0.2-80 ng/ml, e.g., about 0.4-70 ng/ml. e.g., about 0.5-60 ng/ml, e.g.,about 0.8-50 ng/ml, e.g., between about 1 ng/ml to about 40 ng/ml, e.g.,about 1-20 ng/ml, e.g., about 2-20 ng/ml, e.g., about 3-20 ng/ml, e.g.,about 4-15 ng/ml. e.g., about 10 ng/ml.

According to some embodiments of the invention, the synthetic agonist ofthe FGF signaling is C19-jun.

According to some embodiments of the invention, the C19-jun is providedat a concentration of about 1 ng/ml to about 50 ng/ml, e.g., in therange of 10-20 ng/ml.

According to some embodiments of the invention, the transferrin isprovided at a concentration of about 0.1 ng/ml to about 55 μg/ml, e.g.,from about 10 ng/ml to about 10 μg/ml, e.g., between 1-10 μg/ml, e.g.,5.5 μg/ml transferrin.

According to some embodiments of the invention, the selenium is providedat a concentration of about 0.1 ng/ml to about 6000 μg/ml. For example,in order to support fibroblast cell growth the selenium can be providedat a concentration of about 1-10 ng/ml (e.g., about 5 ng/ml of seleniumto support cell growth). Alternatively, to induce adipogenesis from afibroblast cell the selenium can be used at higher concentrations suchas 200-1000 μg/ml, e.g., about 500-800 μg/ml, e.g., about 600 μg/ml toinduce adipogenesis from a fibroblast cell.

The epidermal growth factor superfamily of proteins act as potentmitogenic factors that play an important role in the growth,proliferation and differentiation of numerous cell types. EGF can bepurchased from Peprotech (IL, e.g., Catalogue Number AF10015).

According to some embodiments of the invention, the epidermal growthfactor (EGF) is provided at a concentration of 0.1-30 ng/ml, e.g.,0.5-20 ng/ml, e.g., 1-10 ng/ml, e.g., about 5 ng/ml.

According to some embodiments of the invention, the substitute of EGFcomprises an EGF-R agonist.

According to some embodiments of the invention, the EGF-R agonistcomprises NSC-228155.

According to some embodiments of the invention, the NSC-228155 isprovided at a concentration of about 1 ng/ml to about 100 ng/ml, e.g.,about 5-50 ng/ml.

According to some embodiments of the invention, the Prostaglandin E2(PGE2) is provided at a concentration of 0.01 nM-10 μM, e.g., from about0.1 nM to about 1 μM, e.g., from about 10 nM to about 0.5 μM, e.g., fromabout 50 μM to about 0.5 μM, e.g., about 0.01 μM.

Any of the proteinaceous factors used by the method of some embodimentsof the invention (e.g., the insulin, bFGF, EGF, PGE2) can berecombinantly expressed or biochemically synthesized. In addition,naturally occurring proteinaceous factors such as bFGF can be purifiedfrom biological samples (e.g., from human serum, cell cultures) usingmethods well known in the art. It should be noted that for thepreparation of an animal contaminant-free culture medium theproteinaceous factor is preferably purified from a human source or isrecombinantly expressed.

Biochemical synthesis of the proteinaceous factors of the presentinvention (e.g., the insulin, bFGF, EGF, PGE2) can be performed usingstandard solid phase techniques. These methods include exclusive solidphase synthesis, partial solid phase synthesis methods, fragmentcondensation and classical solution synthesis.

Recombinant expression of the proteinaceous factors of the presentinvention can be generated using recombinant techniques such asdescribed by Bitter et al., (1987) Methods in Enzymol. 153:516-544,Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al.(1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311,Coruzzi et al. (1984) EMBO J. 3:1671-1680, Brogli et al., (1984) Science224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 andWeissbach & Weissbach, 1988, Methods for Plant Molecular Biology,Academic Press, NY, Section VIII, pp 421-463.

Methods of synthesizing the fatty acids, small molecules such asThiazolidinediones (TZD) are known in the art.

According to some embodiments of the invention, the method is performedin-vitro.

Thus, the method of some embodiments of the invention result in theconversion of a fibroblast cell to an adipocyte cells.

Without being bound by any theory, the conversion may occur bytransdifferentiation.

The adipocyte which is formed by the in-vitro method of some embodimentsof the invention, by culturing the spontaneously immortalized fibroblastexhibit the characteristics of a naturally-occurring adipocyte, e.g.,having a compact shape (not elongated), stains positive with Oil-O-Red,and exhibits lipid droplets with a neutral lipid stain (e.g., as shownin FIGS. 4A-D).

According to an aspect of some embodiments of the invention there isprovided an adipocyte cell which is obtainable according to the methodof some embodiments of the invention.

According to an aspect of some embodiments of the invention there isprovided a method of generating a cultured fat on a protein matrix,comprising generating the adipocyte cell generated from the fibroblastaccording to the method of some embodiments of the invention, whereinthe culturing is performed on a plant-derived protein matrix, therebygenerating the cultured fat on the protein matrix.

According to some embodiments of the invention, the plant-derivedprotein matrix is from the legume (Fabaceae) family, from the cerealfamily or from the pseudocereal family.

According to some embodiments of the invention, the plant-derivedprotein matrix is from the legume, Fabaceae, family such as alfalfa,peas, beans, lentils, carob, soybeans, peanuts.

According to some embodiments of the invention, the plant-derivedprotein matrix is from the cereal family such as maize, rice, wheat,barley, sorghum, millet, oats, rye, tritcale, fonio.

According to some embodiments of the invention, the plant-derivedprotein matrix is selected the pseudocereal family including buckwheat,quinoa, or chia

According to some embodiments of the invention, the plant-derivedprotein matrix comprises a soy protein or a pea protein.

According to some embodiments of the invention, the plant-derivedprotein matrix is from a soy protein or a pea protein.

According to an aspect of some embodiments of the invention there isprovided a cultured fat in a plant-derived protein matrix.

According to some embodiments of the invention, the cultured fat in theplant-derived protein matrix includes about 1-1000 million cells pergram.

According to some embodiments of the invention, the cultured fat of someembodiments of the invention is obtainable by the method of someembodiments of the invention.

According to an aspect of some embodiments of the invention there isprovided an in-vitro method of generating a myocyte from a fibroblast,comprising upregulating expression within a spontaneously immortalizedfibroblast of a polypeptide selected from the group consisting of myoD1and myogenin.

Methods of upregulating a level of expression and/or activity of apolypeptide are well known in the art and include recombinant DNAtechniques and/or genome editing methods as is further describedhereinunder.

According to some embodiments of the invention, the upregulation is ofthe myoD1 and myogenin polypeptides.

According to some embodiments of the invention, the chicken myoD1polypeptide is encoded by a polynucleotide comprising the nucleic acidsequence set forth by SEQ ID NO:5.

According to some embodiments of the invention, the chicken myogeninpolypeptide is encoded by a polynucleotide comprising the nucleic acidsequence set forth by SEQ ID NO:7.

According to some embodiments of the invention, the chicken myoD1polypeptide is encoded by the nucleic acid construct set forth by SEQ IDNO: 1 or 3.

According to some embodiments of the invention, the chicken myogeninpolypeptide is encoded by the nucleic acid construct set forth by SEQ IDNO: 2.

According to an aspect of some embodiments of the invention there isprovided a myocyte obtainable according to the methods of someembodiments of the invention.

According to an aspect of some embodiments of the invention there isprovided an in-vitro method of screening for a small molecule capable ofproducing a myocyte, comprising:

(a) transfecting a spontaneously immortalized fibroblast with a nucleicacid construct comprising a nucleic acid sequence encoding a reporterpolypeptide under a transcriptional control of a promoter specificallyactive in myocytes,

(b) contacting a transfected fibroblast resultant of step (a) with atleast one small molecule of a plurality of small molecules, and

(c) detecting activity of the reporter polypeptide above apre-determined threshold in the transfected fibroblast following step(b), wherein presence of the activity above the pre-determined thresholdis indicative that the at least one small molecule is capable ofconverting the spontaneously immortalized fibroblast into the myocyte.

According to some embodiments of the invention, the fibroblast is anavian fibroblast.

According to some embodiments of the invention, the avian is selectedfrom the group consisting of: chicken, duck, goose, and quail.

Non-limiting examples of reporter polypeptides include, the greenfluorescent protein (GFP), blue fluorescent protein (BFP), redfluorescent protein (RFP) or yellow fluorescent protein (YFP).

According to some embodiments of the invention the reporter polypeptideis the COP-GFP (e.g., as shown in FIG. 8). For example, the codingsequence of the COP-GFP can be the nucleic acid sequence set forth bySEQ ID NO: 12.

Fluorescence detection methods which can be used to detect the reporterpolypeptide include for example, fluorescence activated flow cytometry(FACS), immunofluorescence confocal microscopy, fluorescence in-situhybridization (FISH) and fluorescence resonance energy transfer (FRET).

It should be noted that the spontaneously immortalized fibroblasts canbe also used in screening without genetic modification (e.g., visuallyfor instance), for example with an antibody or a dye.

According to an aspect of some embodiments of the invention there isprovided an n-vitro method of generating an edible meat, comprisingculturing:

(a) a spontaneously immortalized fibroblast in a serum-free medium underconditions suitable for converting the fibroblast into an adipocyte,and/or

(b) a spontaneously immortalized fibroblast in a serum-free medium underconditions suitable for converting the fibroblast into a myocyte,

thereby generating the edible meat.

According to an aspect of some embodiments of the invention there isprovided an in-vitro method of generating an edible meat, comprisingculturing:

(a) a spontaneously immortalized fibroblast in a serum-free medium underconditions suitable for converting the fibroblast into an adipocyte,and; or

(b) a spontaneously immortalized fibroblast in a serum-free medium underconditions suitable for converting the fibroblast into a myocyte,

(c) an endothelial cell,

thereby generating the edible meat.

According to some embodiments of the invention, the step (a) and step(b) are effected simultaneously in the same culture system.

According to some embodiments of the invention, the step (a) and step(b) are effected in two distinct (e.g., separated) culture systems.

According to some embodiments of the invention, the steps (a), (b) and(c) are effected simultaneously in the same culture system.

According to some embodiments of the invention, the serum-free mediumcomprises oleic acid and a PPAR-gamma agonist.

According to some embodiments of the invention, the endothelial cell isa spontaneously immortalized endothelial cell.

According to some embodiments of the invention, the endothelial cell isnon-genetically modified.

According to some embodiments of the invention, the culturing isperformed on a scaffold.

According to some embodiments of the invention, the cells attach to thescaffold.

Non-limiting examples of scaffolds include, but are not limited tovarious sponges, matrices, hydrogels or beads;

Examples of suitable sponges include, but are not limited to, polylacticacid, polyglycolic acid, or poly(lactic-co-glycolic acid) (PLGA, SigmaP2191, P2066, P1941, 430471, 764868, 790214, 900289), Variotis™(Biometic, AU), Cellusponge™ (hydroxypropyl cellulose. Bio-ByblosCatalogue No. Z741057).

According to some embodiments of the invention, the scaffold isbiodegradable.

According to some embodiments of the invention, the culturing isperformed in a perfusion system.

According to some embodiments of the invention, the culturing isperformed in the perfusion system of some embodiments of the invention.

According to some embodiments of the invention, the culturing isperformed on an edible hollow fiber cartridge, where nutrient supply ishomogenously distributed in the absence of an integrated vascularnetwork. For example, the fibers of the cartridge are made from ediblenatural or synthetic polymers, such as cellulose (FiberCell, #C3008),cellulose acetate and the cells form a mass surrounding the fibers.Cellulose is FDA approved as GRAS, and used to control moisture andstabilizer shredded cheese, bread, and various sauces.

According to some embodiments of the invention, the culturing isperformed on a vegetable-derived matrix.

According to some embodiments of the invention, the vegetable-derivedmatrix is from a cereal, gluten, or legume.

According to some embodiments of the invention, the vegetable-derivedmatrix is selected from the legume, Fabaceae, family, such as alfalfa,peas, beans, lentils, carob, soybeans, peanuts; or from the cerealfamily, such as maize, rice, wheat, barley, sorghum, millet, oats, rye,tritcale, fonio; and/or from the pseudocereal family includingbuckwheat, quinoa, or chia.

According to some embodiments of the invention, the legume is soy orpea.

According to some embodiments of the invention, the culturing isperformed in a suspension culture devoid of substrate adherence, withoutany adherence of the cells to the scaffold, matrix, sponge, or anycarrier such as micro-carrier beads.

According to an aspect of some embodiments of the invention there isprovided an edible meat obtainable from the method of some embodimentsof the invention.

According to some embodiments of the invention, the edible meat is in aform of a patty of nugget with a density in the range of about 100×10⁶cells/gram to about 500×10⁶ cells/gram, e.g., about 200×10⁶ cells/gram.

According to an aspect of some embodiments of the invention there isprovided a method of generating a spontaneously immortalized fibroblast,comprising:

(a) culturing avian embryo cells in the presence of a serum-containingmedium under adherent culture conditions to thereby obtain chickenembryonic fibroblasts,

(b) passaging the avian embryonic fibroblasts for at least 10-12passages in the serum-containing medium under the adherent conditionsuntil culture collapse, wherein the culture collapse is characterized bysenescence and/or death of at least 90% of avian embryonic fibroblasts,

(c) isolating at least one colony which survived the culture collapse inthe serum-containing medium for at least additional 20 passages,

thereby generating the spontaneously immortalized fibroblast.

As used herein the phrase “culture collapse” refers to a cell culture inwhich the majority of the cells have undergone senescence (i.e., stopcell division) or cell apoptosis/necrosis.

According to some embodiments of the invention, the serum-containingmedium is a DMEM/F12 based medium.

According to some embodiments of the invention, the serum in the mediumcomprises 15% FBS (fetal bovine serum).

According to some embodiments of the invention, the serum-containingmedium further comprises L-Analyl-L-Glutamine.

According to some embodiments of the invention, the chicken embryo isobtained from a fertilized broiler chicken egg grown for 10-12 days.

According to an aspect of some embodiments of the invention there isprovided a spontaneously immortalized chicken fibroblast obtainable bythe method of some embodiments of the invention.

According to some embodiments of the invention, the spontaneouslyimmortalized chicken fibroblast is capable of a continuous passaging forat least about 15, about 20, about 25, about 30, about 35, about 40passages.

According to some embodiments of the invention, the spontaneouslyimmortalized chicken fibroblast is capable of at least about 40, about45, about 50, about 55, about 60, about 65, about 70, about 75, about80, about 85, about 90 or more population doublings.

Upregulation of myoD1 and/or myogenin in a cell (e.g., a spontaneouslyimmortalized fibroblast) can be effected at the genomic level (i.e.,activation of transcription via promoters, enhancers, regulatoryelements), at the transcript level (i.e., correct splicing,polyadenylation, activation of translation) or at the protein level(i.e., post-translational modifications, interaction with substrates andthe like).

Following is a list of agents capable of upregulating the expressionlevel and/or activity of myoD1 and/or myogenin.

An agent capable of upregulating expression of a myoD1 and/or myogeninmay be an exogenous polynucleotide sequence designed and constructed toexpress at least a functional portion of the myoD1 and/or myogenin.Accordingly, the exogenous polynucleotide sequence may be a DNA or RNAsequence encoding a myoD1 and/or myogenin molecule, capable ofconverting the fibroblast to a myocyte cell.

To express exogenous myoD1 and/or myogenin in avian cells, apolynucleotide sequence encoding myoD1 and/or myogenin is preferablyligated into a nucleic acid construct suitable for avian cellexpression. Such a nucleic acid construct includes a promoter sequencefor directing transcription of the polynucleotide sequence in the cellin a constitutive or inducible manner.

It will be appreciated that the nucleic acid construct of someembodiments of the invention can also utilize myoD1 and/or myogeninhomologues which exhibit the desired activity (e.g., capable ofconverting the fibroblast to a myocyte cell). Such homologues can be,for example, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical to SEQ ID NO:5 or 7, as determined using theBestFit software of the Wisconsin sequence analysis package, utilizingthe Smith and Waterman algorithm, where gap weight equals 50, lengthweight equals 3, average match equals 10 and average mismatch equals −9.

Constitutive promoters suitable for use with some embodiments of theinvention are promoter sequences which are active under mostenvironmental conditions and most types of cells such as thecytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoterssuitable for use with some embodiments of the invention include forexample the tetracycline-inducible promoter (Zabala M, et al., CancerRes. 2004, 64(8): 2799-804).

The nucleic acid construct (also referred to herein as an “expressionvector”) of some embodiments of the invention includes additionalsequences which render this vector suitable for replication andintegration in prokaryotes, eukaryotes, or preferably both (e.g.,shuttle vectors). In addition, a typical cloning vectors may alsocontain a transcription and translation initiation sequence,transcription and translation terminator and a polyadenylation signal.By way of example, such constructs will typically include a 5′ LTR, atRNA binding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof.

The nucleic acid construct of some embodiments of the inventiontypically includes a signal sequence for secretion of the peptide from ahost cell in which it is placed. Preferably the signal sequence for thispurpose is a mammalian signal sequence or the signal sequence of thepolypeptide variants of some embodiments of the invention.

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of someembodiments of the invention is active in the specific cell populationtransformed.

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for some embodiments of the inventioninclude those derived from polyoma virus, human or murinecytomegalovirus (CMV), the long term repeat from various retrovirusessuch as murine leukemia virus, murine or Rous sarcoma virus and HIV.See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of myoD1 and/or myogenin mRNAtranslation. Two distinct sequence elements are required for accurateand efficient polyadenylation: GU or U rich sequences located downstreamfrom the polyadenylation site and a highly conserved sequence of sixnucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination andpolyadenylation signals that are suitable for some embodiments of theinvention include those derived from SV40.

In addition to the elements already described, the expression vector ofsome embodiments of the invention may typically contain otherspecialized elements intended to increase the level of expression ofcloned nucleic acids or to facilitate the identification of cells thatcarry the recombinant DNA. For example, a number of animal virusescontain DNA sequences that promote the extra chromosomal replication ofthe viral genome in permissive cell types. Plasmids bearing these viralreplicons are replicated episomally as long as the appropriate factorsare provided by genes either carried on the plasmid or with the genomeof the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

The expression vector of some embodiments of the invention can furtherinclude additional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide.

It will be appreciated that the individual elements comprised in theexpression vector can be arranged in a variety of configurations. Forexample, enhancer elements, promoters and the like, and even thepolynucleotide sequence(s) encoding a myoD1 and/or myogenin can bearranged in a “head-to-tail” configuration, may be present as aninverted complement, or in a complementary configuration, as ananti-parallel strand. While such variety of configuration is more likelyto occur with non-coding elements of the expression vector, alternativeconfigurations of the coding sequence within the expression vector arealso envisioned.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

As described above, viruses are very specialized infectious agents thathave evolved, in many cases, to elude host defense mechanisms.Typically, viruses infect and propagate in specific cell types. Thetargeting specificity of viral vectors utilizes its natural specificityto specifically target predetermined cell types and thereby introduce arecombinant gene into the infected cell. Thus, the type of vector usedby some embodiments of the invention will depend on the cell typetransformed. The ability to select suitable vectors according to thecell type transformed is well within the capabilities of the ordinaryskilled artisan and as such no general description of selectionconsideration is provided herein. For example, bone marrow cells can betargeted using the human T cell leukemia virus type I (HTLV-I) andkidney cells may be targeted using the heterologous promoter present inthe baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) asdescribed in Liang C Y et al., 2004 (Arch Virol. 149: 51-60).

Recombinant viral vectors are useful for in vivo expression of myoD1and/or myogenin since they offer advantages such as lateral infectionand targeting specificity. Lateral infection is inherent in the lifecycle of, for example, retrovirus and is the process by which a singleinfected cell produces many progeny virions that bud off and infectneighboring cells. The result is that a large area becomes rapidlyinfected, most of which was not initially infected by the original viralparticles. This is in contrast to vertical-type of infection in whichthe infectious agent spreads only through daughter progeny. Viralvectors can also be produced that are unable to spread laterally. Thischaracteristic can be useful if the desired purpose is to introduce aspecified gene into only a localized number of targeted cells.

Various methods can be used to introduce the expression vector of someembodiments of the invention into stem cells. Such methods are generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press,Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, AnnArbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al.,Cancer Investigation, 14(1): 54-65 (1996)]. The most preferredconstructs for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral construct suchas a retroviral construct includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger.Such vector constructs also include a packaging signal, long terminalrepeats (LTRs) or portions thereof, and positive and negative strandprimer binding sites appropriate to the virus used, unless it is alreadypresent in the viral construct. In addition, such a construct typicallyincludes a signal sequence for secretion of the peptide from a host cellin which it is placed. Preferably the signal sequence for this purposeis a mammalian signal sequence or the signal sequence of the polypeptidevariants of some embodiments of the invention. Optionally, the constructmay also include a signal that directs polyadenylation, as well as oneor more restriction sites and a translation termination sequence. By wayof example, such constructs will typically include a 5′ LTR, a tRNAbinding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof. Other vectors can be usedthat are non-viral, such as cationic lipids, polylysine, and dendrimers.

Other than containing the necessary elements for the transcription andtranslation of the inserted coding sequence, the expression construct ofsome embodiments of the invention can also include sequences engineeredto enhance stability, production, purification, yield or toxicity of theexpressed peptide. For example, the expression of a fusion protein or acleavable fusion protein comprising the myoD1 and/or myogenin protein ofsome embodiments of the invention and a heterologous protein can beengineered. Such a fusion protein can be designed so that the fusionprotein can be readily isolated by affinity chromatography; e.g., byimmobilization on a column specific for the heterologous protein. Wherea cleavage site is engineered between the myoD1 and/or myogenin proteinand the heterologous protein, the myoD1 and/or myogenin protein can bereleased from the chromatographic column by treatment with anappropriate enzyme or agent that disrupts the cleavage site [e.g., seeBooth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990)J. Biol. Chem. 265:15854-15859].

As mentioned hereinabove, a variety of prokaryotic or eukaryotic cellscan be used as host-expression systems to express the polypeptides ofsome embodiments of the invention. These include, but are not limitedto, microorganisms, such as bacteria transformed with a recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorcontaining the coding sequence; yeast transformed with recombinant yeastexpression vectors containing the coding sequence; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors, such as Ti plasmid, containingthe coding sequence. Mammalian expression systems can also be used toexpress the polypeptides of some embodiments of the invention.

Recovery of the recombinant polypeptide is effected following anappropriate time in culture. The phrase “recovering the recombinantpolypeptide” refers to collecting the whole fermentation mediumcontaining the polypeptide and need not imply additional steps ofseparation or purification. Not withstanding the above, polypeptides ofsome embodiments of the invention can be purified using a variety ofstandard protein purification techniques, such as, but not limited to,affinity chromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

An agent capable of upregulating a myoD1 and/or myogenin in a cell mayalso be any compound which is capable of increasing the transcriptionand/or translation of an endogenous DNA or mRNA encoding the myoD1and/or myogenin and thus increasing endogenous myoD1 and/or myogeninactivity.

According to some embodiments of the invention, over-expression of thepolypeptide of the invention is achieved by means of genome editingusing methods well known in the art.

Genome editing is a powerful mean to impact target traits bymodifications of the target plant genome sequence. Such modificationscan result in new or modified alleles or regulatory elements. Thus,genome editing employs reverse genetics by artificially engineerednucleases to cut and create specific double-stranded breaks at a desiredlocation(s) in the genome, which are then repaired by cellularendogenous processes such as, homology directed repair (HDR) andnon-homologous end-joining (NHEJ). NHEJ directly joins the DNA ends in adouble-stranded break, while HDR utilizes a homologous sequence as atemplate for regenerating the missing DNA sequence at the break point.In order to introduce specific nucleotide modifications to the genomicDNA, a DNA repair template containing the desired sequence must bepresent during HDR. Genome editing cannot be performed using traditionalrestriction endonucleases since most restriction enzymes recognize a fewbase pairs on the DNA as their target and the probability is very highthat the recognized base pair combination will be found in manylocations across the genome resulting in multiple cuts not limited to adesired location. To overcome this challenge and create site-specificsingle- or double-stranded breaks, several distinct classes of nucleaseshave been discovered and bioengineered to date. These include themeganucleases, Zinc finger nucleases (ZFNs), transcription-activatorlike effector nucleases (TALENs) and CRISPR/Cas system.

Over expression of a polypeptide by genome editing can be achieved by:(i) replacing an endogenous sequence encoding the polypeptide ofinterest or a regulatory sequence under the control which it is placed,and/or (ii) inserting a new gene encoding the polypeptide of interest ina targeted region of the genome, and/or (iii) introducing pointmutations which result in up-regulation of the gene encoding thepolypeptide of interest (e.g., by altering the regulatory sequences suchas promoter, enhancers, 5′-UTR and/or 3′-UTR, or mutations in the codingsequence).

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B”, “A or B”, “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

When reference is made to particular sequence listings, such referenceis to be understood to also encompass sequences that substantiallycorrespond to its complementary sequence as including minor sequencevariations, resulting from, e.g., sequencing errors, cloning errors, orother alterations resulting in base substitution, base deletion or baseaddition, provided that the frequency of such variations is less than 1in 50 nucleotides, alternatively, less than 1 in 100 nucleotides,alternatively, less than 1 in 200 nucleotides, alternatively, less than1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides,alternatively, less than 1 in 5,000 nucleotides, alternatively, lessthan 1 in 10,000 nucleotides.

It is understood that any Sequence Identification Number (SEQ ID NO)disclosed in the instant application can refer to either a DNA sequenceor a RNA sequence, depending on the context where that SEQ ID NO ismentioned, even if that SEQ ID NO is expressed only in a DNA sequenceformat or a RNA sequence format. For example, SEQ ID NO: 5 is expressedin a DNA sequence format (e.g., reciting T for thymine), but it canrefer to either a DNA sequence that corresponds to a MyoD1 nucleic acidsequence, or the RNA sequence of an RNA molecule nucleic acid sequence.Similarly, though some sequences are expressed in a RNA sequence format(e.g., reciting U for uracil), depending on the actual type of moleculebeing described, it can refer to either the sequence of a RNA moleculecomprising a dsRNA, or the sequence of a DNA molecule that correspondsto the RNA sequence shown. In any event, both DNA and RNA moleculeshaving the sequences disclosed with any substitutes are envisioned.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

The system of the present embodiments can be used for other purposes aswell. For example, in an embodiment of the present invention, the systemcan be used to generate human tissue from human cell lines with thescope of transplantations. The cells can be autologous, allologous orheterologous with respect to the patient. The procedures described abovecan be used in the manufacturing of partial or full organs fortransplantation.

Numbered Clauses

-   Clause 1: Some embodiments of the present invention provide a system    for growing cells comprising: a primary tissue perfusion circuit    comprising: a tissue growth chamber; at least one first pump; a    culture medium perfusate; an oxygenator; and a heating element; a    secondary dialysis circuit comprising: at least one second pump; a    dialyzer; and a dialysate; where the order of each component in each    circuit of the system can be in any order.-   Clause 2: Some embodiments of the present invention provide the    system where the tissue growth chamber is a jacketed tissue growth    chamber.-   Clause 3: Some embodiments of the present invention provide the    system where the tissue growth chamber is characterized by having a    volume and internal dimensions that are configured and arranged to    receive the growing tissue and a sufficient amount of the culture    medium perfusate to continuously circulate the culture medium    perfusate through the growing tissue.-   Clause 4: Some embodiments of the present invention provide the    system where the first, second or third pump are selected from a    group consisting of peristaltic pump, positive displacement pump,    impulse pump, velocity pump, gravity pump, steam pump, valveless    pumps, and any combination thereof.-   Clause 5: Some embodiments of the present invention provide the    system where the culture medium perfusate comprises non-animal    serum.-   Clause 6: Some embodiments of the present invention provide the    system where the culture medium perfusate comprises components    selected from the group consisting of: amino acids, vitamins, trace    elements, transferrin, insulin, plant-derived recombinant albumin,    bacteria-derived recombinant albumin, tryptose phosphate, glutamine,    glucose, fructose, sucrose, M199, DMEM/F12 medium, KO-serum,    linoleic acid, oleic acid, palmate acid, lecithin, bFGF, IGF-1,    Insulin, SCF, EGF, TGFβ1, IL-11, PGE, BMP4, activin A,    hydrocortisone, ascorbic acid, and any combination thereof.-   Clause 7: Some embodiments of the present invention provide the    system where the oxygenator is a membrane oxygenator.-   Clause 8: Some embodiments of the present invention provide the    system where the oxygenator is adapted to provide at least one gas    selected from the group consisting of oxygen (O₂), carbon dioxide    (CO₂), nitrogen (N₂) and any combination thereof.-   Clause 9: Some embodiments of the present invention provide the    system where the oxygenator is adapted to maintain a    ratio:percentage of each gas of O₂ from about 21% to about 95%, CO₂    from about 0% to about 10% and N₂ from about 0% to about 80%, inside    the system.-   Clause 10: Some embodiments of the present invention provide the    system where the oxygenator is adapted to maintain a    ratio:percentage of each gas of O₂ at about 80%, CO₂ at about 5% and    N₂ at about 15%, inside the system.-   Clause 11: Some embodiments of the present invention provide the    system where the system further comprises a bubble trap.-   Clause 12: Some embodiments of the present invention provide the    system where the bubble trap is equally interchangeable with a    debubbler or a hybrid bubble trap/debubbler.-   Clause 13: Some embodiments of the present invention provide the    system where the heating element is a heat exchanger.-   Clause 14: Some embodiments of the present invention provide the    system where the heating element is selected from the group    consisting of: shell and tube heat exchanger, plate heat exchanger,    plate and shell heat exchanger, adiabatic wheel heat exchanger,    plate fin heat exchanger, pillow plate heat exchanger, fluid heat    exchanger, waste heat recovery units, dynamic scraped surface heat    exchanger, phase-change heat exchanger, direct contact heat    exchanger, microchannel heat exchanger, helical-coil heat exchanger,    spiral heat exchanger, and any combination thereof.-   Clause 15: Some embodiments of the present invention provide the    system where the oxygenator and the heating element are two distinct    components.-   Clause 16: Some embodiments of the present invention provide the    system where the oxygenator and the heating element are one    component.-   Clause 17: Some embodiments of the present invention provide the    system where the dialyzer comprises a membrane having a pore size    selected from a range of 1 to 60 kDa.-   Clause 18: Some embodiments of the present invention provide the    system where the dialyzer comprises a membrane having an area    selected from the range of 10 to 10000 cm².-   Clause 19: Some embodiments of the present invention provide the    system where the system further comprises at least one carbon    filter.-   Clause 20: Some embodiments of the present invention provide the    system where the at least one carbon filter is adapted to clean    toxins present in the dialysate.-   Clause 21: Some embodiments of the present invention provide the    system where the at least one ammonia filter is adapted to clean    ammonia present in the dialysate.-   Clause 22: Some embodiments of the present invention provide the    system where toxins and ammonia are removed by the same filter.-   Clause 23: Some embodiments of the present invention provide the    system where the dialysate comprises glucose, amino acids, insulin,    hormones such as cortisone, and growth factors in serum-free medium.-   Clause 24: Some embodiments of the present invention provide the    system further comprising at least one sensor selected from the    group consisting of temperature sensor, pH sensor, volume sensor,    flow sensor, optical sensor, glucose sensor, oxygen sensor, weight    sensor, protein sensor and any combination thereof.-   Clause 25: Some embodiments of the present invention provide the    system further comprising at least one computer comprising at least    one non-transitory computer readable medium, the non-transitory    computer-readable medium storing a program that causes the computer    to execute a method using a processor that executes the stored    program.-   Clause 26: Some embodiments of the present invention provide the    system where the computer is connected to at least one remote server    allowing to an external remote operator to access the computer.-   Clause 27: Some embodiments of the present invention provide the    system where the program allows the system to operate automatically    without the need of an external operator.-   Clause 28: Some embodiments of the present invention provide the    system where the tissue growth chamber is adapted to grow tissue    originated from cells selected from a group consisting of: primary    cells, embryonic/neonatal fibroblasts cells, embryonic/neonatal    endothelium cells, embryonic/neonatal muscle cells, pluripotent stem    cells, embryonic stem cells, induced pluripotent stem cells (iPSC),    mesenchymal stem cells, fibroblasts cells, endothelial cells,    myocyte cells, satellite cells, hepatocyte cells, blood cells,    neuron cells, fat cells, and any combination thereof.-   Clause 29: Some embodiments of the present invention provide the    system where the cells are exposed to small molecule-based    reprogramming.-   Clause 30: Some embodiments of the present invention provide the    system where the small molecules are selected but not limited to a    group consisting of: CHIR9902, SB431542, RepSox, Parnate, Forskolin,    TTNPB, DZnep, VPA, CHIR99021, PD0325901, PD173074, LIF, A83-01,    BIX01294, AS8351, SC1, Y27632, OAC2, SU16F, JNJ10198409, LDN193189,    NSC 228155, CN 009543V, AG1478, PD 153035, 2-Me-5HT, D4476, RG108,    BIO, SMI1, SMI2, 5-azacytidine, phenamil, GW7845, RG14620, or    Harmine, thiazolidinediones (i.e. rosiglitazone, pioglitazone,    lobeglitazone), IBMX, and any combination thereof.-   Clause 31: Some embodiments of the present invention provide the    system where the cells may stably comprise an inducible controlled    expression transgene system or similar constructs in their genome.-   Clause 32: Some embodiments of the present invention provide the    system where the inducible controlled expression transgene system is    a TET-on or TET-off system.-   Clause 33: Some embodiments of the present invention provide the    system where the induced controlled transgene expressed is MyoD.-   Clause 34: Some embodiments of the present invention provide the    system where the inducible controlled expression transgene system is    activated or deactivated by Doxycycline or similar    activators/deactivators.-   Clause 35: Some embodiments of the present invention provide the    system where the cells are grown in a biodegradable scaffold    contained in the closed-loop perfusion circuit.-   Clause 36: Some embodiments of the present invention provide the    system where the cells are from a non-human animal source selected    from the group consisting of: chicken, turkey, duck, quail, goose,    dove, pheasant, ostrich, cow (calf), deer, goat, sheep (lamb),    horse, lama, camel, rabbit, kangaroo, alligator, turtle, lobster,    salmon, tuna, dolphin, whale and any combination or related species    thereof.-   Clause 37: Some embodiments of the present invention provide the    system where the system is used to grow cells, tissue, partial or    full organs from human or animal origin for transplantation    purposes.-   Clause 38: It is hence a scope of the present invention to provide a    method for growing cells comprising: acquiring a primary tissue    perfusion circuit comprising: a tissue growth chamber; at least one    first pump; a culture medium perfusate; an oxygenator; and a heating    element; acquiring a secondary dialysis circuit comprising: at least    one second pump; a dialyzer; and a dialysate; connecting the primary    tissue perfusion circuit with the secondary dialysis circuit;    growing the cells in the tissue growth chamber until reaching the    desired quantity.-   Clause 39: Some embodiments of the present invention provide the    method where the tissue growth chamber is a jacketed tissue growth    chamber.-   Clause 40: Some embodiments of the present invention provide the    method where the tissue growth chamber is characterized by having a    volume and internal dimensions that are configured and arranged to    receive the growing tissue and a sufficient amount of the culture    medium perfusate to continuously circulate the culture medium    perfusate through the growing tissue.-   Clause 41: Some embodiments of the present invention provide the    method where the first, second or third pump are selected from a    group consisting of peristaltic pump, positive displacement pump,    impulse pump, velocity pump, gravity pump, steam pump, valveless    pumps, and any combination thereof.-   Clause 42: Some embodiments of the present invention provide the    method where the culture medium perfusate comprises non-animal    serum.-   Clause 43: Some embodiments of the present invention provide the    method where the culture medium perfusate comprises components    selected from the group consisting of: amino acids, vitamins, trace    elements, transferrin, insulin, plant-derived recombinant albumin,    bacteria-derived recombinant albumin, tryptose phosphate, glutamine,    glucose, fructose, sucrose, M199, DMEM/F12 medium, KO-serum,    linoleic acid, oleic acid, palmate acid, lecithin, bFGF, IGF-1,    Insulin, SCF, EGF, TGFβ1, IL-11, BMP4, PGE, activin A,    hydrocortisone, ascorbic acid, and any combination thereof.-   Clause 44: Some embodiments of the present invention provide the    method where the oxygenator is a membrane oxygenator.-   Clause 45: Some embodiments of the present invention provide the    method where the oxygenator is adapted to provide at least one gas    selected from the group consisting of oxygen (O₂), carbon dioxide    (CO₂), nitrogen (N₂) and any combination thereof.-   Clause 46: Some embodiments of the present invention provide the    method where the oxygenator is adapted to maintain a    ratio:percentage of each gas of O₂ from about 21% to about 95%, CO₂    from about 0% to about 10% and N₂ from about 0% to about 80%, inside    the system.-   Clause 47: Some embodiments of the present invention provide the    method where the oxygenator is adapted to maintain a    ratio:percentage of each gas of O₂ at about 80%, CO₂ at about 5% and    N₂ at about 15%, inside the system.-   Clause 48: Some embodiments of the present invention provide the    method where the system further comprises a bubble trap.-   Clause 49: Some embodiments of the present invention provide the    method where the bubble trap is equally interchangeable with a    debubbler or a hybrid bubble trap/debubbler.-   Clause 50: Some embodiments of the present invention provide the    method where the heating element is a heat exchanger.-   Clause 51: Some embodiments of the present invention provide the    method where the heating element is selected from the group    consisting of: shell and tube heat exchanger, plate heat exchanger,    plate and shell heat exchanger, adiabatic wheel heat exchanger,    plate fin heat exchanger, pillow plate heat exchanger, fluid heat    exchanger, waste heat recovery units, dynamic scraped surface heat    exchanger, phase-change heat exchanger, direct contact heat    exchanger, microchannel heat exchanger, helical-coil heat exchanger,    spiral heat exchanger, and any combination thereof.-   Clause 52: Some embodiments of the present invention provide the    method where the oxygenator and the heating element are two distinct    components.-   Clause 53: Some embodiments of the present invention provide the    method where the oxygenator and the heating element are one    component.-   Clause 54: Some embodiments of the present invention provide the    method where the dialyzer comprises a membrane having a pore size    selected from a range of 1 to 300 kDa.-   Clause 55: Some embodiments of the present invention provide the    method where the dialyzer comprises a membrane having an area    selected from the range of 10 to 10000 cm².-   Clause 56: Some embodiments of the present invention provide the    method where the system further comprises at least one carbon    filter.-   Clause 57: Some embodiments of the present invention provide the    method where the at least one carbon filter is adapted to clean    toxins present in the dialysate.-   Clause 58: Some embodiments of the present invention provide the    method where the dialysate comprises glucose, amino acids, insulin,    hormones such as cortisone, and growth factors in serum-free medium.-   Clause 59: Some embodiments of the present invention provide the    method further comprising at least one sensor selected from the    group consisting of temperature sensor, pH sensor, volume sensor,    flow sensor, optical sensor, glucose sensor, oxygen sensor, weight    sensor, protein sensor and any combination thereof.-   Clause 60: Some embodiments of the present invention provide the    method further comprising at least one computer comprising at least    one non-transitory computer readable medium, the non-transitory    computer-readable medium storing a program that causes the computer    to execute a method using a processor that executes the stored    program.-   Clause 61: Some embodiments of the present invention provide the    method where the computer is connected to at least one remote server    allowing to an external remote operator to access the computer.-   Clause 62: Some embodiments of the present invention provide the    method where the program allows the system to operate automatically    without the need of an external operator.-   Clause 63: Some embodiments of the present invention provide the    method where the tissue growth chamber is adapted to grow tissue    originated from cells selected from a group consisting of: primary    cells, embryonic/neonatal fibroblasts cells, embryonic/neonatal    endothelium cells, embryonic/neonatal muscle cells, pluripotent stem    cells, embryonic stem cells, induced pluripotent stem cells (iPSC),    mesenchymal stem cells, fibroblasts cells, endothelial cells,    myocyte cells, satellite cells, hepatocyte cells, blood cells,    neuron cells, fat cells, and any combination thereof.-   Clause 64: Some embodiments of the present invention provide the    method where the cells are exposed to small molecule-based    reprogramming.-   Clause 65: Some embodiments of the present invention provide the    method where the small molecules are selected but not limited to a    group consisting of: CHIR9902, SB431542, RepSox, Parnate, Forskolin,    TTNPB, DZnep, VPA, CHIR99021, PD0325901, PD173074, LIF, A83-01,    BIX01294, AS8351, SC1, Y27632, OAC2, SU16F, JNJ10198409, LDN193189,    NSC 228155, CN 009543V, AG1478, PD 153035, 2-Me-5HT, D4476, RG108,    BIO, SMI1, SMI2, 5-azacytidine and any combination thereof.-   Clause 66: Some embodiments of the present invention provide the    method where the cells may stably comprise an inducible controlled    expression transgene system or similar constructs in their genome.-   Clause 67: Some embodiments of the present invention provide the    method where the inducible controlled expression transgene system is    a TET-on or TET-off system.-   Clause 68: Some embodiments of the present invention provide the    method where the induced controlled transgene expressed is MyoD.-   Clause 69: Some embodiments of the present invention provide the    method where the inducible controlled expression transgene system is    activated or deactivated by Doxycycline or similar    activators/deactivators.-   Clause 70: Some embodiments of the present invention provide the    method where the cells are grown in a biodegradable scaffold    contained in the closed-loop perfusion circuit.-   Clause 71: Some embodiments of the present invention provide the    method where the cells are from a non-human animal source selected    from the group consisting of: chicken, turkey, duck, quail, goose,    dove, pheasant, ostrich, cow (calf), deer, goat, sheep (lamb),    horse, lama, camel, rabbit, kangaroo, alligator, turtle, lobster,    salmon, tuna, dolphin, whale and any combination or related species    thereof.-   Clause 72: It is hence a scope of some embodiments of the present    invention to grow cells wherein the cells are grown in a system as    described herein.-   Clause 73: Some embodiments of the present invention provide the    edible in-vitro meat, where the in-vitro meat is grown in the    presence of components selected from the group consisting of: amino    acids, vitamins, trace elements, transferrin, insulin, plant-derived    recombinant albumin, bacteria-derived recombinant albumin, tryptose    phosphate, glutamine, glucose, fructose, sucrose, M199 medium,    KO-serum, linoleic acid, oleic acid, palmate acid, lecithin, bFGF,    IGF-1, SCF, EGF, TGFβ1, IL-11, BMP4, activin A, hydrocortisone,    ascorbic acid, and any combination thereof.-   Clause 74: Some embodiments of the present invention provide the    edible in-vitro meat, where the in-vitro meat is grown in an    environment characterized by a ratio:percentage of each gas of O₂    from about 21% to about 95%, CO₂ from about 0% to about 10% and N₂    from about 0% to about 80%, inside the system.-   Clause 75: Some embodiments of the present invention provide the    edible in-vitro meat, where the in-vitro meat is grown in an    environment characterized by a ratio:percentage of each gas of O₂ at    about 80%, CO₂ at about 5% and N₂ at about 15%, inside the system.-   Clause 76: Some embodiments of the present invention provide the    edible in-vitro meat, where the in-vitro meat is originated from    cells selected from a group consisting of: primary cells,    embryonic/neonatal fibroblasts cells, embryonic/neonatal endothelium    cells, embryonic/neonatal muscle cells, pluripotent stem cells,    embryonic stem cells, induced pluripotent stem cells (iPSC),    mesenchymal stem cells, fibroblasts cells, endothelial cells,    myocyte cells, satellite cells, hepatocyte cells, blood cells,    neuron cells, fat cells, and any combination thereof.-   Clause 77: Some embodiments of the present invention provide the    edible in-vitro meat, where the cells are exposed to small    molecule-based reprogramming.-   Clause 78: Some embodiments of the present invention provide the    edible in-vitro meat, where the small molecules are selected but not    limited to a group consisting of: CHIR9902, SB431542, RepSox,    Parnate, Forskolin, TTNPB, DZnep, VPA, CHIR99021, PD0325901,    PD173074, LIF, A83-01, BIX01294, AS8351, SC1, Y27632, OAC2, SU16F,    JNJ10198409, LDN193189, NSC 228155, CN 009543V, AG1478, PD 153035,    2-Me-5HT, D4476, RG108, BIO, SMI1, SMI2, 5-azacytidine and any    combination thereof.-   Clause 79: Some embodiments of the present invention provide the    edible in-vitro meat, where the cells may stably comprise an    inducible controlled expression transgene system or similar    constructs in their genome.-   Clause 80: Some embodiments of the present invention provide the    edible in-vitro meat, where the inducible controlled expression    transgene system is a TET-on or TET-off system.-   Clause 81: Some embodiments of the present invention provide the    edible in-vitro meat, where the induced controlled transgene    expressed is MyoD.-   Clause 82: Some embodiments of the present invention provide the    edible in-vitro meat, where the inducible controlled expression    transgene system is activated or deactivated by Doxycycline or    similar activators/deactivators.-   Clause 83: Some embodiments of the present invention provide the    edible in-vitro meat, where the cells are grown in a biodegradable    scaffold.-   Clause 84: Some embodiments of the present invention provide the    edible in-vitro meat, where the cells are from a non-human animal    source selected from the group consisting of: chicken, turkey, duck,    quail, goose, dove, pheasant, ostrich, cow (calf), deer, goat, sheep    (lamb), horse, lama, camel, rabbit, kangaroo, alligator, turtle,    lobster, salmon, tuna, dolphin, whale and any combination or related    species thereof.-   Clause 85: Some embodiments of the present invention provide the    edible in-vitro meat, where the cells are grown in a non-animal    serum medium.-   Clause 86: Some embodiments of the present invention provide the    edible in-vitro meat, where the cells are grown in a medium which    comprises glucose, amino acids, insulin, hormones such as cortisone,    and growth factors in serum-free medium.-   Clause 87: It is hence a scope of the present invention to provide a    transplantable in-vitro tissue wherein the in-vitro tissue is    manufactured in a system as described herein.-   Clause 88: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the in-vitro tissue is grown in    the presence of components selected from the group consisting of:    amino acids, vitamins, trace elements, transferrin, insulin,    plant-derived recombinant albumin, bacteria-derived recombinant    albumin, tryptose phosphate, glutamine, glucose, fructose, sucrose,    M199 medium, KO-serum, linoleic acid, oleic acid, palmate acid,    lecithin, bFGF, IGF-1, SCF, EGF, TGFβ1, IL-11, BMP4, activin A,    hydrocortisone, ascorbic acid, and any combination thereof Clause    89: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the in-vitro meat is grown in    an environment characterized by a ratio:percentage of each gas of O₂    from about 21% to about 95%, CO₂ from about 0% to about 10% and N₂    from about 0% to about 80%, inside the system.-   Clause 90: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the in-vitro meat is grown in    an environment characterized by a ratio:percentage of each gas of O₂    at about 80%, CO₂ at about 5% and N₂ at about 15%, inside the    system.-   Clause 91: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the tissue is originated from    cells selected from a group consisting of: primary cells,    embryonic/neonatal fibroblasts cells, embryonic/neonatal endothelium    cells, embryonic/neonatal muscle cells, pluripotent stem cells,    embryonic stem cells, induced pluripotent stem cells (iPSC),    mesenchymal stem cells, fibroblasts cells, endothelial cells,    myocyte cells, satellite cells, hepatocyte cells, blood cells,    neuron cells, fat cells, and any combination thereof.-   Clause 92: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the cells are exposed to small    molecule-based reprogramming.-   Clause 93: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the small molecules are    selected but not limited to a group consisting of: CHIR9902,    SB431542, RepSox, Parnate, Forskolin, TTNPB, DZnep, VPA, CHIR99021,    PD0325901, PD173074, LIF, A83-01, BIX01294, AS8351, SC1, Y27632,    OAC2, SU16F, JNJ10198409, LDN193189, NSC 228155, CN 009543V, AG1478,    PD 153035, 2-Me-5HT, D4476, RG108, BIO, SMI1, SMI2, 5-azacytidine    and any combination thereof.-   Clause 94: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the cells may stably comprise    an inducible controlled expression transgene system or similar    constructs in their genome.-   Clause 95: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the inducible controlled    expression transgene system is a TET-on or TET-off system.-   Clause 96: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the induced controlled    transgene expressed is MyoD.-   Clause 97: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the inducible controlled    expression transgene system is activated or deactivated by    Doxycycline or similar activators/deactivators.-   Clause 98: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the cells are grown in a    biodegradable scaffold.-   Clause 99: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the cells are from a non-human    animal source selected from the group consisting of: chicken,    turkey, duck, quail, goose, dove, pheasant, ostrich, cow (calf),    deer, goat, sheep (lamb), horse, lama, camel, rabbit, kangaroo,    alligator, turtle, lobster, salmon, tuna, dolphin, whale and any    combination or related species thereof.-   Clause 100: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the cells are from a human    source.-   Clause 101: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the cells are grown in a    non-animal serum medium.-   Clause 102: Some embodiments of the present invention provide the    transplantable in-vitro tissue where the cells are grown in a medium    which comprises glucose, amino acids, insulin, hormones such as    cortisone, and growth factors in serum-free medium.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below findexperimental, and/or calculated support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Closed-Loop Perfusion Circuit for Growth of Chicken Liver

Liver is a highly nutritious, high value product, with relatively softconsistency due to the low abundance of fibrillar matrix and hive-likestructure. A genetic modification is used to induce the proliferation ofchicken hepatocytes and endothelial cells, allowing to optimize theclose-loop perfusion circuit for high-density tissue growth. Closed-loopperfusion includes, as mentioned above, a dialysis unit permittingphysiological addition of nutrients and removal of toxins, instead ofcomplete media replacement.

1.1 Closed-Loop Perfusion Circuit The perfusion system is composed of aprimary tissue perfusion circuit and a secondary dialysis circuit fornutrient and toxin exchange (10). The primary circuit includes culturemedium perfusate that is recirculated using a peristaltic pump through ajacketed tissue growth chamber, a membrane oxygenator, a heat exchanger,and a bubble trap. The oxygenator is gassed with a mixture of 80% O₂/5%CO_(2/15)% N₂ maintaining constant pH.

A fraction of the perfusate is diverted to secondary circuit through aSpectrum Labs hollow fiber dialyzer (Rancho Dominguez, Calif.) with a790 cm² membrane area and a 30 kDa molecular weight cutoff at a rate of3 mL/min/gram tissue. The secondary circuit dialyzed the perfusate bycounter-current exposure to protein-free dialysate, recirculated througha carbon filter using a third peristaltic pump. Temperature within thesystem is maintained at 37° C.

The main advantage of dialysis is that albumin, with a molecular weightof 66.5 kDa, is retained in the main perfusion circuit. Albumin has ahalf-life of 20 days and is a carrier protein of growth factors,peptides (e.g. insulin), and fatty acids. Albumin and growth factors arethe main cost driver of culture medium.

1.2 Model Cells and Tissue Growth

Recently, it has been demonstrated that expression of E6/E7 proteinspermitted the rapid expiation of functional human hepatocytes, liverendothelial and stellate cells under OSM-stimulation (1). Stablyinfected E6/E7LOW hepatocytes with GFP, endothelial cells with mCherry,and stellate cells with CFP using lentivirus reporters were used foroptimization of the device. These fluorescent markers helped assesstissue organization and proliferation rates before beginning the actualproduction of the chicken liver. Seeded on a soft hydrogel matrix, thiscell mixture rapidly forms a proliferating liver organoid (11).

Cells are mixed in ratio of 1:1:0.1 for hepatocytes, endothelial cells,and stellate cells, respectively, spun down and re-suspended in 0.1 mlhydrogel matrix composed of animal-free synthetic polypeptides with poresize of 50 to 200 nm (Sigma, A6982). Hydrogel-cell suspension areinjected into a biodegradable polymer scaffold with pore size of 50 to1000 μm, and placed in the jacked tissue growth chamber. While thehydrogel polypeptides will be replaced with native extracellular matrixwithin 5-7 days, the polymer scaffold will support the growing tissuefor 14-28 days until it reaches significant mass and the cells cannot bewashed away.

1.3 Growth Optimization

To optimize tissue growth and minimization of nutrient addition, tissueuptake rates of glucose, glutamine, fatty acids and albumin werecarefully analyzed by present inventor aiming to keep consternationsconstant. Perfusate and dialysate were automatically sampled usingmicrofluidic switchboard every 4 hours to monitor glucose, lactate,glutamine, fatty acid and albumin content (12). Oxygen content wasmeasured dynamically using optical sensors (13).

Data was used to determine rate and volume of medium supplementation asa function of tissue growth rate.

Tissue morphology and growth rates were quantified daily using confocalmicroscopy. Human albumin and bile acid production were measured in theperfusate every 24 hours, marking liver-specific function. Finally, theabsence of necrosis or apoptosis was assessed using H&E and TUNELstaining following 7, 14 and 28 days of growth.

Example 2 Development of Small Molecule-Based Expansion of Serum-FreeCultures of Chicken Hepatocytes, Endothelial Cells and Fibroblasts

Chemical compounds offer an attractive alternative to growth factorsthat are generally used to stimulate serum-free cell growth. Smallmolecules are far less expensive than recombinant growth factors, havelower lot-to-lot variability, are non-immunogenic and are much morestable.

2.1 Developing and Optimizing Minimal Growth Medium

Chicken fibroblasts were purchased from Charles River Laboratories(Wilmington, Mass.) and expanded in serum-free medium composed of M199supplemented with 0.5 mg/mL plant-derived albumin (Cellastim™), 0.6 μMlinoleic and oleic acid, 0.6 μg/mL soy lecithin, 7.5 mML-Alanyl-L-Glutamine, 0.1 μM dexamethasone, 50 μg/mL ascorbic acid, and0.5 U/mL insulin (Eli Lilly). This minimal medium was generally furthersupplemented with 5 ng/mL bFGF, 5 ng/mL EGF, and 30 pg/mL TGFβ1 tosupport 4-fold faster proliferation of fibroblasts, at considerableexpense. Cells were expanded in complete medium up to PD (populationdoubling) 15 to generate frozen stocks, and were used between PD 15 and25. To study whether all growth factors were essential for chickenfibroblast expansion, the present inventor assessed proliferation ratesas a function of growth factor concentration aiming to find a minimalcombination.

Chicken embryonic endothelial cells were isolated from fertilized eggsor purchased from Charles River Laboratories (Wilmington, Mass.). Cellswere expanded in serum-free endothelium medium composed of RPMI1640supplemented with 3.75 mg/mL plant-derived albumin (Cellastim™), 0.6 μMlinoleic and oleic acid, 0.6 μg/mL soy lecithin, 7.5 mML-Alanyl-L-Glutamine, 0.1 μM dexamethasone, 50 μg/mL ascorbic acid, and0.5 U/mL insulin (Eli Lilly). This minimal medium was furthersupplemented with 5 ng/mL bFGF, 5 ng/mL EGF, and 10 ng/mL VEGF. Cellswere expanded in complete medium up to PD 5 to generate frozen stocks,and are used between PD 5 and 10. To study whether all growth factorswere essential for chicken endothelial cell expansion, the presentinventor assessed proliferation rates as a function of growth factorconcentration aiming to find a minimal combination.

Chicken embryonic muscle cells were isolated from fertilized eggs orpurchased from Charles River Laboratories (Wilmington, Mass.). Cellswere expanded in serum-free medium composed of M199 supplemented with0.5 mg/mL plant-derived albumin (Cellastim™), 0.6 μM linoleic and oleicacid, 0.6 μg/mL soy lecithin, 7.5 mM L-Alanyl-L-Glutamine, 0.1 μMdexamethasone, 50 μg/mL ascorbic acid, and 0.5 U/mL insulin (Eli Lilly).This minimal medium was generally further supplemented with 5 ng/mLbFGF, 5 ng/mL EGF, and 30 pg/mL IGF-1. Cells were expanded in completemedium up to PD 15 to generate frozen stocks, and were used between PD 5and 10. To study whether all growth factors were essential for chickenmyocytes expansion, the present inventor assessed proliferation rates asa function of growth factor concentration aiming to find a minimalcombination.

Chicken hepatocytes were purchased from Charles River Laboratories(Wilmington, Mass.) and seeded in serum-free formulation composed ofWilliams E medium supplemented with 3.75 mg/mL plant-derived albumin(Cellastim™), 0.2 μM linoleic and oleic acids, 2 mML-Alanyl-L-Glutamine, 0.1 μM dexamethasone, 5 μg/mL transferrin, and 0.5U/mL insulin (Eli Lilly). It has been shown that this serum-free mediumsupports the robust expansion of genetically modified human hepatocytes(1). Cells were exposed to FPH1 (BRD-6125), FPH2 (BRD-9424), and FH1(BRD-K4477) small molecules identified to enhance proliferation ofunmodified human hepatocytes (7). The present inventor expected thatlimited proliferation would be achieved due to evolutionary conservationof liver regeneration signaling pathways. High throughput screen ofchicken hepatocytes without small-molecule driven expansion is stillpossible, but it is simply more expensive.

2.2 Identification of Small Molecule Growth Enhancers in a HighThroughput Screen

High content screening of small molecules is carried out at the BroadInstitute of MIT and Harvard or an equivalent robotic screeningfacility. A separate screening for chicken fibroblasts, endothelialcells, myocytes and hepatocytes is carried out. Cells are seeded in384-well screening plates (Corning) at a density of 10,000 cells/cm² inthe appropriate minimal growth medium without supplements. Plates areincubated at 37° C. and 5% CO₂ and medium is replaced daily. A libraryof 12,480 compounds is added at concentration of 15 μM and incubated for48 hours. The present inventor carries out a standard MTT analysis;acquired phase images of the treated cells, and Hoechst analysis fortotal DNA. To identify functional proliferation hits, the positive MTTand DNA increase are integrated based on p-value.

Chemicals producing functional proliferation hits were combined in asmaller screening profile aiming to identify minimal functionalcombinations that produce the greatest fold increase in proliferation.Based on earlier reports (7), the present inventor expected 2 to 3 smallmolecules to be identified in each screen. For example, NSC-228155 wasrecently shown to be an EGF-R agonist (16). Once small moleculescocktails were identified, the present inventor attempted to add backgrowth factors at lower concentrations to see if greater proliferationenhancement can be achieved in a cost-efficient manner.

Example 3 Development of Small-Molecule Based Differentiation of ChickenMuscle Cells

Myocyte expansion is usually limited to 15 population doublings,producing 16-gram tissue from each isolate. However, myocytes can bedifferentiated from pluripotent stem cells in a multistep processmimicking myogenesis (17). Alternatively, fibroblasts can be convertedto myocytes using MyoD expression (18) or a cocktail of small molecules(8).

Pluripotent stem cells double every 44±13 hours and their serum freemedium costs about $540/liter. In contrast, fibroblasts double every21±3 hours and their serum free medium costs about $272/liter. Thismeans that using current techniques, pluripotent stem cells will produce1 kg tissue after 39 days, at $100,000/kg, while fibroblasts will do soafter 18 days, at $50,000/kg. Therefore, the present inventor' approachprimarily focused on genetic and chemical differentiation of fibroblaststo myocytes, with pluripotent stem cells studied to mitigate risk.

3.1 Generation of Tetracycline-Dependent MyoD Expressing ChickenFibroblasts

Doxycycline (Dox) is an analog of tetracycline that can be used torapidly activate gene expression by binding a reversetetracycline-controlled advanced transactivator (rtTA2^(S)-M2) that actson a tetracycline responsive element (TRE). Dox shows no apparenttoxicity, is inexpensive and can be readily washed out of the cellsfollowing activation. The system has been shown to reliably work onchicken embryos (19).

The present inventor has generated a stable line of chicken fibroblastsexpressing Dox-inducible chicken MyoD, by introducingpCAGGS-rtTA2^(S)-M2 and pTRE-MyoD plasmids under puromycin selection.Chicken fibroblasts were exposed to 0.5 ng/μl Dox for 48 hour and MyoDexpression was evaluated by qRT-PCR. Conversion to myocytes wasevaluated 7 and 12 days after Dox induction by staining for myosin heavychain (MyHC) and titin (18). Dox-induced muscle cells served as positivecontrol and a genetically engineered (GE) alternative to smallmolecule-induced conversion of fibroblasts to myocytes.

3.2 Identification of Small-Molecule Cocktail for Conversion of Myocytes

Recently mouse fibroblasts were converted to cardiomyocytes by atwo-step combination of small molecules promoting reprogramming;including CHIR9902, RepSox, Forskolin, and VPA followed by 2i (CHIR99021and PD0325901) conditions promoting myocardium development; includingCHIR99021, PD0325901, and LIF (9). Human fibroblasts were similarlyconverted using a combination of reprogramming anddifferentiation-inducing factors CHIR99021, A83-01, BIX01294, AS8351,SC1, Y27632, OAC2, SU16F and JNJ10198409 (8). Conversion was slow,taking 20 to 30 days and producing about 6% cardiomyocytes.

In a screen of zebrafish, mouse, and human cells, Xu (Xu et al. Cell155, 909-921, 2013) and colleagues identified 6 small molecules thatexpanded muscle progenitors, including the adenylyl cyclase activator,forskolin. A combination of bFGF, forskolin, and the GSK3b inhibitor BIOinduced skeletal muscle differentiation of human induced pluripotentstem cells (21). In a different screen, a group identified SMI1 and SMI2to robustly induce skeletal muscle differentiation from pluripotent stemcells, while others showed 5-azacytidine can similarly promotemyogenesis (22). These results suggest that a two-step procedure totrans-differentiate skeletal muscle using a reprogramming cocktail (6),followed factors that promote skeletal muscle myogenesis in pluripotentstem cells, can produce promising results.

The present inventor' approach was to stably transfect chickenfibroblasts with EGFP reporter for MyHC for high throughput screening(8). Cells were exposed to varying cocktails of reprogramming andmyogenic factors discussed above, as well as those identified in example2.2. Cells were evaluated based on EGFP fluorescence and myofibermorphology after 20 days of induction. To identify functional hits, thepresent inventor integrated positive MyHC and morphology hits based onp-value.

3.3. Developing Direct Differentiation of Chicken Pluripotent Stem Cells

The avian embryo spends only 20 hours in utero as it descends down theoviduct. By the time the egg is laid, the epiblast is a single layercomprised of 20,000-50,000 cells. Chicken embryonic stem cells arederived from this blastodisc and can be perpetuated in culture,producing all somatic lineages but not the germline (3). Like mouseembryonic stem cells, they require LIF to remain undifferentiated.Culture medium includes bFGF, IGF-1, SCF, and IL-11, in addition to LIF(23).

Recently, serum-free protocols for differentiation of muscle fibers werepublished for mouse and human pluripotent stem cells (17). Mouse stemcells were induced toward a mesoderm phenotype in N2B27 mediumcontaining 10 ng/ml BMP4 for 2 days, DMEM medium containing 15% knockoutserum, 0.5% DMSO, 0.1 μM LDN193189, and 1 μM CHIR99021 for 4 days. Thenmesodermal cells differentiated to skeletal muscle in DMEM mediumcontaining 15% knockout serum, 10 ng/ml HGF, 2 ng/ml IGF-1, 20 ng/mlbFGF, and 0.1 μM LDN193189 for 8 days. The protocol is robust,generating 30-60% muscle cells in 14 days.

As noted above, other groups identified additional small molecules thatdrive the differentiation of pluripotent stem cells toward skeletalmuscle cells. These include the combination of bFGF, forskolin and BIO(21), SMI1 and SMI2 (24), and finally 5-azacytidine (22).

The present inventor' approach was to translate existing serum-freemouse protocols to chicken embryonic stem cells taking into accountdifferences in avian development pathways (25). Small moleculesidentified in previous studies were used to augment differentiation andincrease muscle fiber density.

Example 4 Establishing Closed-Loop Perfusion Circuit for Growth andDifferentiation of Chicken Muscle

Muscle tissue is highly packed myofiber cluster nourished by endothelialcapillaries. Fibrillar collagen, secreted by the mesenchyme plays asignificant role in tissue consistency. The present inventor's approachwas to grow a high density of chicken fibroblasts and endothelial cellsin a biodegradable scaffold contained in a closed-loop perfusion circuitoptimized in example 1. Shear forces helped align collagen fibersdeposited by the growing fibroblasts. Once sufficient mass was reached,small molecules were introduced in differentiation medium convertingfibroblasts to skeletal muscle cells (example 3) and allowing themyofiber to align along to shear-aligned fibers.

Closed-loop perfusion included a dialysis unit permitting physiologicaladdition of nutrients and removal of toxins, instead of complete mediareplacement. The main advantage of dialysis was that albumin, with amolecular weight of 66.5 kDa, was retained in the main perfusioncircuit. Albumin has a half-life of 20 days and is a carrier protein ofgrowth factors and fatty acids. Albumin and growth factors are the maincost drivers of culture medium.

4.1 Closed-Loop Perfusion Circuit

The perfusion system that was optimized in example 1.1 was used here.Briefly, the primary circuit included culture medium perfusate that wasrecirculated using a peristaltic pump through a jacketed tissue growthchamber, a membrane oxygenator (80% 02, 5% CO₂, and 15% N₂), a heatexchanger (37° C.), and a bubble trap. A fraction of the perfusate wasdiverted to a hollow fiber dialyzer with a 2200 cm² membrane area and a30 kDa molecular weight cutoff at a rate of 3 mL/min/gram tissue. Thesecondary circuit dialyzed the perfusate by counter-current exposure toprotein-free dialysate and recirculated through a carbon filter using asecond peristaltic pump.

4.2 Model Cells and Tissue Growth

Cell seeding that was optimized in example 1.2 was used here. Theexperiment used a mixture of Dox-MyoD chicken fibroblasts developed inexample 3.1 and endothelial cells at 10:1 ratio. Briefly, cells weresuspended in 0.1 ml hydrogel matrix composed of animal-free syntheticpolypeptides with pore size of 50 to 200 nm (Beaver Labs). Hydrogel-cellsuspension was injected into a biodegradable polymer scaffold with poresize of 50 to 1000 μm, and placed in the jacked tissue growth chamber.While the hydrogel polypeptides were replaced with native extracellularmatrix within 5-7 days, the polymer scaffold supported the growingtissue for 14 days until it reached significant mass and the cells couldnot be washed away. Scaffold was removed at 5 and 10 days fixed andsectioned for analysis. The present inventor stained for collagen type-Ideposition and alignment, and analyzed connective tissue density andhealth using H&E staining.

The present inventor introduced 0.5 ng/μl Dox for 4 days, inducingconversion of fibroblasts to muscle cells. Then Dox was washed out for 4days, replaced with IFG-1 to promote cell fusion to muscle fibers.Tissue was removed at day 14 and 18 fixed and sections for analysis. Thepresent inventor stained for MyHC, desmin and titin, and analyzed theresulting muscle tissue density and health using H&E staining. Comparingdesmin and MyHC positive cells, as well as qRT-PCR assessed the degreeof muscle formation.

4.3 Growth Optimization

Tissue growth was optimized to adjust the feeding parameters to thegrowing cells and the differentiation method used. Tissue uptake ratesof glucose, glutamine, fatty acids and albumin were analyzed aiming tokeep consternations constant. Perfusate and dialysate were automaticallysampled using microfluidic switchboard every 4 hours to monitor glucose,lactate, glutamine, fatty acid and albumin content (12). Oxygen contentwas measured dynamically using optical sensors (13). Data was used todetermine rate and volume of medium supplementation as a function oftissue growth rate.

Tissue growth rates were quantified using AlamarBlue® (Thermo FisherSci.) a non-toxic, secreted, cell viability indicator. Finally, theabsence of necrosis or apoptosis was assessed using H&E and TUNELstaining following 6, 12 and 18 days of growth.

4.4 Cell-Specific Approach

Some embodiments of the present intention are to reach 150 grams ofchicken muscle tissue in each circuit, equivalent to a large drumstickor chicken breast. This represents a mass of 3×10¹⁰ cells achieved inabout 18 population doublings.

For fibroblasts, it represents 16 to 18 days of growth. MyoD-inducedconversion is rapid (18) allowing to grow fibroblasts for 10 days anddifferentiate them for 8 days in culture. In contrast, small moleculebased reprogramming approaches (8) (9) are reported to take between 24to 30 days, at least for cardiomyocytes. While the developmentallysimpler skeletal muscle differentiation will undoubtedly be shorter,insights from example 3.2 played a critical role in deciding the initialseeding densities and the timing of conversion.

Importantly, this cell density represents 33 to 35 days of growth forembryonic stem cells. The current serum-free differentiation protocol(17) requires only 14 days of differentiation. Therefore, chickenembryonic stem cells can be seeded at higher densities and grown inpluripotency medium for 19 days. The main challenge for embryonic stemcells growth is that the tissue cannot be endothelialized as endothelialcells would promote differentiation. This means that individualembryonic stem cell clusters must be smaller than 0.5 mm in diameter, orsuffer necrosis at the core. One solution was to seed embryonic stemcells on biodegradable alginate microparticles (Quad Technologies)allowing the suspension to grow separately within the tissue growthchamber. The present inventor has previously been successful in growinghuman embryonic stem cells in a similar high-density suspension cultures(26).

Example 5 Chicken-Based Laboratory Grown Meat: Generation ofSpontaneously Immortalized Chicken Fibroblast Cell Line

The following Example illustrates non-limiting cells, which can be usedfor culturing meat in-vitro.

Animal-free, high-density expansion of chicken cells—Various independentcell sources can be used for growing meat in-vitro.

(1) Chicken embryonic fibroblasts were isolated and expanded untilspontaneous immortalization occurred.

(2) An immortal chicken iPSC line is generated using non-integratingvectors or small molecules from which fibroblasts can be obtained byroutine differentiation.

(3) Several established ATCC cell lines can be used. These include DF1(chicken), QM7 (quail), and DE (duck).

(4) Integrating vectors are used to establish chicken iPSC lines asdescribed in literature (Intarapat & Stern 2013).

Derivation of a Spontaneously Immortalized Line of Chicken EmbryonicFibroblasts

Experimental methods—Fertilized broiler chicken eggs were grown at 38.5°C. for 10-12 days in a humidified incubator. Eggs were opened betweenday 10 to 12 and embryos removed. Heads, limbs and internal organs wereremoved, and cells were mechanically extracted and plated on tissueculture treated plastic in DMEM/F12 medium supplemented with 15% FBS(fetal bovine serum), and 2 mM of L-Analyl-L-Glutamine.

Experimental Results—Under these conditions, in the absence of any othergrowth factors, fibroblasts outgrow the culture resulting in homogenouspopulations of primary chicken embryonic fibroblasts (CEFs) (FIGS.2A-B). Roughly 2×10⁷ cells were isolated per embryo, with multiplepopulations cultured in parallel. Initial CEF morphology was elongated,becoming more compact with increasing passage number (FIG. 2B and datanot shown). Most CEF cultures became senescent by population doubling(PD) 30-40 (data not shown); with 2-3 colonies surviving the crisisbecoming spontaneously immortalized chicken fibroblasts (CSIFs; FIG.2C). CSIF show fibroblast morphology and exhibit a doubling time of 18±2hours by PD 90 (FIG. 2E).

Example 6 Chicken-Based Laboratory Grown Meat: Identification of aSerum-Free Medium for Propagating Spontaneously Immortalized ChickenFibroblast Cell Line

Development of Serum-Free Medium for CSIF Propagation—The CSIFs readilygrow on tissue culture plastic in DMEM/F12 medium supplemented with 15%FBS, and 2 mM of L-Analyl-L-Glutamine (FIG. 2D). There are severalserum-free medium formulations for the growth of human and mousefibroblasts, including PCS-201-040 (ATCC) and TheraPEAK (Lonza), bothfailed to support the proliferation of primary CEF or the novel CSIFline obtained by the present inventor (FIGS. 2D, 2E and data not shown).

To develop a serum-free medium that supports the culture of CEF andCSIF, the present inventor formulated a minimal medium composed ofDMEM/F12 supplemented with 0.1 μM dexamethasone, 10 μg/ml insulin, 5.5μg/ml transferrin, and 5 ng/ml selenium (ITS), 12 μM linoleic and 12 μMoleic acids, and 2 mM of L-Analyl-L-Glutamine. Cells were plated in FBScontaining medium, and transferred to minimal medium after overnightattachment. Basal medium was supplemented with growth factors andhormones showing that while heparin and T3 had little effect of CSIFgrowth (data not shown), the addition of basic Fibroblast Growth Factor(bFGF, 10 ng/ml) was essential (FIGS. 3C and 3F), showing 20±2 hoursdoubling time (data not shown). In addition, Epidermal Growth Factor(EGF, 5 ng/ml), Prostaglandin E2 (PGE2, 0.01 μM) and Growth Hormone (GH,10 ng/ml) supported the proliferation of CEF and CSIF (FIGS. 3D, 3E, 3Fand data not shown).

The optimal growth medium tested by the present inventor was composed ofDMEM/F12 supplemented with dexamethasone (0.1 μM), 1×ITS+3 (Sigma,I2771), bFGF (10 ng/ml), EGF (5 ng/ml), and PGE2 (0.01 μM) resulting insimilar growth rates to a culture medium containing 15% FBS.

Under some conditions, insulin could be replaced with IGF-1 (5 ng/ml),or the stabilized Long R3 IGF-1 [Sigma (5 ng/ml)]. EGF can be replacedwith the EGF-R agonist [NSC-228155 (Sakanyan et al. Sci. Reports. 2014]at a concentration of 5-50 ng/ml. FGF can similarly be replaced with asmall molecule or synthetic agonist such as C19-jun (Ballinger et al.Nature. Biotech. 1999) at a concentration of 10-20 ng/ml.

A screen for small molecules is carried out essentially as described inExample 2 above. The first small molecule screen attempts to identifymolecules that can replace growth factors and hormones in the culturemedium (e.g. insulin, FGF, EGF, TGFβ). Thus, a sequential removal of onegrowth factor or hormone at a time is performed, aiming to reach thesame growth rate with a small molecule replacement.

The co-culture of endothelial cells with the fibroblasts allows thepresent inventor to remove some growth factors that are naturallyproduced by the endothelium.

Additionally or alternatively, cells are engineered to specificallyproduce these growth factors, thereby reducing overall cost.

It should be noted that the lack of attachment factors (e.g.vitronectin, fibronectin) in serum-free medium makes it difficult toserially passage CEF or CSIF. Since animal or human derivedextracellular matrix proteins must be avoided other natural, recombinantproteins and/or synthetic polymers such as Poly-D-Lysine can be used topropagate cells in the absence of serum.

Example 7 Chicken-Based Laboratory Grown Meat: Conversion of aSpontaneously Immortalized Chicken Fibroblast Cell Line into AdipocuteIna Serum-Free Medium

Conversion of CSIF to Adipocytes in Serum-free Medium—In mammalianspecies, preadipocytes can be readily differentiated into adipocytesusing 3-isobutyl-1-methylxanthine (IBMX) in the presence of insulin, andcortisone (e.g. dexamethasone). Preadipocytes are seeded at 70%confluence in serum-containing medium supplemented with 0.5 mM IBMX, 0.1μM dexamethasone, and 10 μg/ml insulin for 3 days, followed by 3-daytreatment with insulin alone, which is then removed at day 6 to finalizedifferentiation. The protocol works on primary preadipocyte andpreadipocyte cell lines such as 3T3-L1 and 3T3-F442A, but not onfibroblasts. Recent work identified multiple small molecules that canenhance the differentiation of preadipocytes to adipocytes inserum-containing medium, including PPARg activators: phenamil, GW7845,RG14620, or Harmine (Park et al. J. Lipid Research. 2010; Waki et al.Cell Met. 2007). Clinically approved drugs of the thiazolidinedionefamily (i.e. rosiglitazone, pioglitazone, lobeglitazone) that targetPPARg could potentially have similar effects on preadipocytes.

Chicken preadipocyte have yet to be identified, leading most groups touse stromal-vascular cells derived from chicken adipose tissues(Matsubara et al. Comp. Bio. & Phys 2008). However, IBMX anddexamethasone have no affect on these chicken preadipocytes, whileexposure to 200-400 μM oleic acid induces their differentiation toadipocytes in the presence of serum (Zhouchun et al. Biosci. Rep. 2014;Matsubara et al. Comp. Bio. & Phys 2008).

Efforts to differentiate primary CEF to adipocytes showed that exposureto 400 μM oleic acid and 20% serum induced lipid accumulation in theelongated primary cells (Liu et al. Comp. Bio. & Phys 2009). Themaster's thesis of Aishlin Elizabeth Lee (Ohio State U. 2013) showed asimilar effect in response to 100-300 μg/l of selenium and 2% serum.Both works used primary chicken cells, cultured in the presence ofserum.

To develop a protocol for conversion of the spontaneously immortalizedchicken fibroblasts (CSIF line) under serum-free conditions, the presentinventor seeded the CSIF at 70% confluence in the optimized serum-freeDMEM/F12 medium supplemented with dexamethasone (0.1 μM), 1×ITS+3(Sigma, I2771), and bFGF (10 ng/ml). The CSIF cells were treated for 4or 7 days with 200 to 400 μM oleic acid alone, or in combination with0.5 mM IBMX, or 10 μM of the FDA-approved small molecule Rosiglitazone.While all oleic acid treatments increased lipid accumulation, only theaddition of IBMX or Rosiglitazone supported a rounded adipogenicphenotype (FIGS. 4A-D).

Stimulation of mitochondria proliferation in myocytes—Additionally oralternatively, a dual-PPARα/γ agonist such as naringenin (Goldwasser etal. PLoS One 2010) is used to stimulate mitochondria proliferation inmyocytes, expanding their protein content, and adipogenicdifferentiation of the remaining fibroblasts to fat.

Example 8 Chicken-Based Laboratory Grown Meat: Conversion of aSpontaneously Immortalized Chicken Fibroblast Cell Line into Myocytes

Generation of Dox-inducible MyoD1 and PPARγ vectors—Dox-inducible MyoD1and PPARγ vectors were generated. These vectors are capable oftransforming chicken fibroblasts (primary or immortalized) towardmyocytes and adipocytes with high efficiency, respectively (data notshown).

Previous work showed that expression of the MyoD1 gene is sufficient toinduce myogenesis of human and mouse fibroblasts. A parallel butconnected myogenesis pathway goes through Myogenin (MYOG) in mammals.

Experimental Results

Genetic Conversion of CSIF to Myocytes—To examine if similar conversionof chicken cells to myocytes is possible, the present inventor generatedseveral nucleic acid constructs (vectors) as is schematicallyillustrated in FIGS. 6-8 and 12. The first construct [FIG. 6,“pinducer-VP64-cMyoD1”, SEQ ID NO:3] included the chicken MyoD1 gene(SEQ ID NO:5) cloned into a Dox-inducible lentiviral vector (pInducer20)being fused to the VP64 transcriptional activator (SEQ ID NO: 6) thathas been shown to improve MyoD1 induced differentiation in mouse cells(Kabadi et al. ACS Synthetic Biology. 2015). A second lentiviral vector(SEQ ID NO:2, FIG. 7) was created for Dox-inducible chicken MYOGexpression included the cMyogenin coding sequence (SEQ ID NO: 7) underthe control of the minimal CMV promoter (SEQ ID NO: 8). A thirdlentiviral vector (SEQ ID NO:1, FIG. 12) was created for Dox-induciblechicken MYOD1 expression included the cMyoD1 coding sequence (SEQ ID NO:5) under the control of the minimal CMV promoter (SEQ ID NO: 8). Allthree vectors were sequenced and were found to be mutation free (datanot shown).

To rapidly detect myogenesis in culture the present inventor created aGFP reporter construct (lentiviral reporter construct; SEQ ID NO: 4,FIG. 8) for the rat myosin light chain-3 promoter-enhancer (rMLC3-GFP),including the rat MLC3 enhancer (SEQ ID NO: 10; 1.5 kb enhancer sequencefrom the rat MLC3 gene), the rat MLC3 promoter (SEQ ID NO: 11; 628 bppromoter sequence) and the COP-GFP coding sequence (SEQ ID NO: 12). Inthis lentiviral reporter construct the rat MCL3 enhancer and promoterdriving expression of the COP-GFP. This reporter has been shown to bespecific and effective in chicken embryos and cells (McGrew et al. BMCDevelopmental Biology 2010). The various constructs were introduced into293T cells in order to generate lentivirus.

Primary CEF and CSIF lines were infected 3 times with the lentivirusvectors and split a day later. Cells were cultured in standard DMEM/F12medium containing 15% serum. CEF and CSIF cultures were induced bydoxycycline and were followed for 30 days. While non-induced cultureswere negative for GFP (Data not shown), both CEF and CSIF show strongexpression of MLC3 by day 11 of culture with cells forming distinctfibers maintained to day 30 of culture (FIGS. 5B and 5C).Immunofluorescence analysis showed F-actin organization andmultinucleated (syncytia) fiber formation (FIG. 5D). Staining showedclear induction of α1-skeletal muscle actin (ACTA1) and Troponin T showsa clear muscle phenotype as early as day 7 of induction (FIG. 5E).

As described above in Example 3 hereinabove, a small molecule screenaiming to identify small molecules that can transdifferentiatefibroblasts to muscle cells in the absence of Dox-inducible MyoD1 iscarried out using the a GFP reporter construct (lentiviral reporterconstruct) for the rat myosin light chain-3 promoter-enhancer (FIG. 8).Thus, the present inventor uses variants of small molecule cocktailsrecently shown to transdifferentiate mouse and human fibroblasts tocardiomyocytes (Cao et al 2016; Fu et al. 2015). A GFP-linked MLCreporter ensures a rapid detection of successful conversion as shown inFIGS. 5B-C.

It should be noted that there are regulatory concerns regarding the useof some small molecules that can affect DNA structure in thereprogramming step. Regulatory agencies are already looking at thisissue for human regenerative medicine, while other groups are rapidlyproducing alternative small molecules for conversion. In contrast toregenerative medicine approaches, the perfusion system of someembodiments of the invention can rapidly flush the system and remove anyresidual small molecules before the process terminates. Additionally oralternatively, a Dox-inducible differentiation method can be used asshown in FIGS. 5A-E.

Metabolomic analysis of the perfusate and tissue is carried out overtime to identify which nutrients are rate limiting (i.e. missing). Ametabolic flux balance model of the tissue is established (as describedin Levy et al. 2016) which allows to see changing fluxes and determinethe metabolic requirements of the cells. Growth factors are introducedin access and their removal is determined by protein array analysis, assmall molecules are to replace them. Metabolic analysis using JobstTechnologies (Freiburg, German) metabolic sensors and an oxygen sensor,showed proliferating CSIF consume oxygen at a rate of 2.4 nmol/min/10⁶cells, consume glucose at a rate of 1.8 nmol/min/10⁶ cells and producelactate at a rate of 198 pmol/min/10⁶ cells during the growth phase.

Example 9 Generation of a Hybrid of Plant-Based Meat Substitute Productwith Laboratory Grown Fat

A meat analogue, also called a meat substitute, approximates certainaesthetic qualities (primarily texture, flavor and appearance) orchemical characteristics of specific types of meat. Many analogues arebased on cereal, gluten, or legumes such as soy or pea. Global meatsubstitute market was $3.3 billion in 2014, and grows at a CAGR of 7.5%including products such as veggie burgers, soy hotdogs, and chickennuggets. However, these products fail to emulate the flavor and aroma ofanimal meat. Recent work on plant-based meat substitutes identifiedfermented leghemoglobin (also called “(also leghaemoglobin orlegoglobin”) as a source for a metallic flavor resembling blood. Usingmolecular gastronomy tools companies such as Impossible Foods and BeyondMeat produced ground meat-like patty with the texture and aroma of beef.However, the cooking of protein-bound saturated fat produces thedistinct flavor of meat. Current products use coconut or palm oil as asource of palmitate (16:0) that is solid at room temperature, butrapidly melts at 62.9° C. This results in an oily, dripping product thatis distinct from real beef. Similarly, several companies such as BeyondMeat extrude layered legume protein to create the texture and mouth feelof chicken strips. Similar lack of animal fat results in a dry mouthfeel distinct from real chicken.

To produce animal fat, CSIF are cultured in serum free medium composedof DMEM/F12 supplemented with dexamethasone (0.1 μM), bFGF (10 ng/ml),long IGF-1 (Sigma I1271) (5 ng/ml), 12 μM linoleic acid, and 2 mM ofL-Analyl-L-Glutamine. Cells are cultured in fed-batch bioreactors,perfusion bioreactors, or closed-loop perfusion described above to adensity of 10×10⁶ cells/ml. Medium further supplemented with 400 μMoleic acid and 10 μM rosiglitazone. Cells acquire lipid droplets andreach a density of 100×10⁶ cells/ml. The adipocyte slurry isconcentrated and added as raw material to the plant-based matrixcomposed of cereal or legume-based protein isolate such as the PeaProtein Organic Powder (Now Sports). Raw material density is changed asa function of the desired end product. Chicken strips require 5 to 10%laboratory grown adipocytes, resulting in about 1.5×10⁸ cells in finalproduct. Hamburgers require 10 to 20% laboratory grown adipocytes,resulting in about 3×10⁸ cells in final product.

Example 10 Generation of a Chicken Patty or Nugget in a StirredBioreactor

Culturing of chicken fibroblasts in a stirred bioreactor—Chickenfibroblasts can be cultured in a stirred bioreactor (BioFlo® 320) insmall single use vessels of 250-400 mL volume.

The cells are aggregated into small micro-clusters and are cultured insuspension without micro-carrier beads. This permits the high-densitygrowth of cells reaching 4-6×10⁶ cells/mL. Once this density is reacheda chicken patty or nugget with a density of 200×10⁶ cells/gram isprepared for a public tasting.

Alternatively, fibroblasts can be grown on collagen-coated micro-carrierbeads (e.g. SoloHill Engineering) as previously described (Mg & Ma Sha2015).

Example 11 Establishment and Isolation of Chicken Embryonic EndothelialCells

Differentiation of chicken induced pluripotent stem cells (iPSCs) intoendothelial cells—Using chicken fibroblasts (non-immortalized) thepresent inventor generated chicken induced pluripotent stem cells(iPSCs) essentially as described by Vodyanik et al. 2010. Then thechicken iPSCs are used for the differentiation of chicken endothelialcells in a similar manner to human and mouse derived cells (Giacomelliet al. Development 2017). The iPS-derived chicken endothelial cells canbe used as is with limited population doubling (up to 20) or can be usedto generate spontaneously immortalized endothelial cells as describedbelow.

Spontaneous immortalization of chicken endothelial cells—Chickenmicrovascular endothelial cells which are either obtained from acommercial source (Charles River Labs) or which are isolated from youngchickens according to established protocols (Twal & Leach In Vitro Cell.Dev. Biol. Animal 1996) are then being cultured on 50 μg/ml collagentype I or 0.2% gelatin in standard culture medium, such as EGM2mv(Lonza, Switzerland) or serum free formulation (e.g. ThermoFisher#11111044) containing bFGF (20 ng/ml), EGF (10 ng/ml), and human plasmafibronectin (10 μg/ml) until a spontaneous immortalization occurs, so asto obtain a chicken endothelial cell line which is not geneticallymodified.

It is noted that the present inventor was able to obtain a spontaneouslyimmortalized endothelial cell from rat cardiac microvascular endothelialcells purchased from Vec Technologies (Rensselaer, N.Y.), reaching atleast population doubling 120 (data not shown), thus proving that aspontaneous immortalization of endothelial cells is feasible.

Example 12 Generation of Chicken Muscle Using Sponges

Generation of chicken muscle tissue by co-culturing of spontaneouslyimmortalized fibroblasts and spontaneously immortalized endothelialcells on sponges (scaffolds)—The present inventor has designedgeneration of chicken muscle by seeding spontaneously immortalizedchicken fibroblasts and spontaneously immortalized rat endothelial cellsmixtures into a biodegradable large pore sponges, such as collagenhydrogel, that permits rapid vascularization and uniform distribution ofnutrients. The micro-tissue is characterized by confocal and electronmicroscopy.

Other suitable sponges (scaffolds) include, but are not limited to,polylactic acid, polyglycolic acid, or poly(lactic-co-glycolic acid),sponges, polyglicolic acid sponges, Variotis™ (Biometic, AU) orCellusponge™ (hydroxypropyl cellulose. Bio-Byblos Catalogue No.Z741057).

It is noted that one possible way of avoiding loss of cells by theperfusion system, is to first embed the cells in an injectable hydrogelpolypeptide matrix which is then being injected into the biodegradablesponge.

The micro-tissue scaffold is cultured under perfusion and the cellproliferation and metabolic uptake of nutrients and growth factors wastracked as shown in Table 2 below. Non-specific absorption by the systemis monitored, even in the absence of cells, since this could lead toloss of peptides and lipids.

TABLE 2 Metabolic Flux Measurement Oxygen Consumption Rate  2.4nmol/min/10⁶ cells Glucose Uptake Rate  1.8 nmol/min/10⁶ cells LactateProduction Rate  198 pmol/min/10⁶ cells

Growth rates and metabolic parameters are reintroduced into the modeland systems parameters are adjusted.

Cell growth and the maximal cell density are determined in the absenceof dialysis.

Following the successful demonstration of cell growth under perfusionthe tissue organization and the proper vascular connectivity anddistribution are characterized as shown in FIGS. 11A-C. Spontaneouslyimmortalized chicken fibroblasts (CSIF) and spontaneously immortalizedrat microvascular endothelial cells (RCEC) were suspended at a densityof 150×10⁶ CSIF/ml and 15×10⁶ RCEC/ml in collagen type 1 scaffold andseeding for microscope evaluate and perfusion. High-density tissueformed overnight and compacted the collagen scaffold. As shown bysulforhodamine B stain, the cultured cells revealed high protein content(FIG. 11A). The tissue seeded in the bioreactor were sealed and perfusedin serum free medium, without antibiotics for 11 days. No loss of cellmass was observed. Confocal analysis showed clear organization ofvascular structures and associated tissue.

Growth factors and cytokines are used to define vascular maturation.Tissue assembly and growth are characterized by live imaging and endpoint microscopic evaluation. Once cell density outstrips nutrientuptake, perfusion rate through the nested dialysis circuit is increasedto rapidly remove toxins while adding stable supply of nutrients to thegrowing tissue. The above-described model shows that the minimalperfusion rate required to support cell growth increases exponentiallywith time or linearly with tissue mass to supply the oxygen consumptionrates of the cells. A minimal perfusion rate of 36.9 ml/s is necessaryto sustain 1 kg of tissue in ambient 21% oxygen, but only 8.2 ml/s is berequired if oxygen partial pressure is raised to 95% in the oxygenator.The minimal perfusion rate can decrease by increasing the oxygencarrying capacity of the medium using an oxygen carrier such asperfluorocarbon emulsion (e.g. Fluosol) or modified hemoglobin (e.g.Hemopure). Hemopure® is a hemoglobin-based oxygen carrier manufacturedby HbO2 Therapeutics LLC that has an oxygen carrying capacity of 1.39 mlO2/g Hb, meaning that if we add 3.55 μg of Hemopure per ml of media wedouble the oxygen content, decreasing by 2 the perfusion rate needed toperfuse a large bulk of tissue.

The model also suggests that glucose is not a limiting factor forperfusion, as flow rates under 0.4 ml/sec can deliver sufficient glucoseto over 1 kg of cells. However, as glucose if is not replenished, 1 kgof tissue will consume all glucose in the system within 48 minutes. Atotal of 140 grams of glucose are required for tissue growth. Glucoseonly becomes limiting when tissue passes 24 grams in mass, and will needto be added at hourly intervals on the final two days of growth.

Alternatively, tissue growth is explored in edible hollow fibercartridge, where nutrient supply is homogenously distributed in theabsence of an integrated vascular network.

Here, the fibers of the cartridge are made from edible natural orsynthetic polymers, such as cellulose (FiberCell, #C3008), and the cellsform a mass surrounding the fibers. Cellulose is FDA approved as GRAS,and used to control moisture and stabilizer shredded cheese, bread, andvarious sauces.

Example 13

A prototype system as been designed, according to some embodiments ofthe present invention. The prototype system is illustrated schematicallyin FIG. 9, and is based on a closed loop dialysis bioreactor. The corecircuit is a recirculating perfusion bioreactor, 1 to 5 liters involume, that grows muscle tissue growing from 20 mg to 1000 grams, andthat retains cells using a hollow fiber cartridge, packed bed design, orvascularized embedded tissue configuration. An increasing percentage ofthe bioreactor outflow is circulated through a counter flow dialysis,whose pores are designed to exclude albumin, about 30 kDa molecularweight cutoff. As the cells are not present during this filtration stepit can occur at high pressures. This design retains the albumin and withit the growth factors and lipids is carries in the medium. Anotherperfusion circulates the dialysate through a filter that removes ammoniaand toxins (e.g. Zeolite molecular sieve). This design can reach thevolume/mass ratio of animals, nominally 100 ml per kg mass. It isestimated that about 2 liters medium can be used with per 5 litersbioreactor volume. This design can produce 2.5 kg mass every 10 days,consuming only 2 liters of medium, as it does not require a seed train.This translates to $4 per kg mass for the medium costs alone.

Capital costs are also considered. Current estimate of the bill of partsusing off the shelf components is about $7,000, suggesting manufacturingcosts of about $300 for a system having a 5 liter bioreactor chamber. Aproduction facility with 5,000 such systems can cost about $1.5 million,so that an estimate of about $5 million for the entire facility.Assuming the same 10% annual depreciation and maintenance costs, aproduction capacity of about 450,000 kg/year is obtained with about$500,000 annual costs to maintain. This results in a capital cost ofonly about $1.1 per kg mass produced.

The prototype system is composed of a primary tissue perfusion circuitand a secondary dialysis circuit for nutrient and toxin exchange. Theprimary circuit includes culture medium perfusate that is recirculatedusing a peristaltic pump through a jacketed tissue growth chamber, anoxygenator, a heat exchanger, and a bubble trap. The oxygenator isgassed with a mixture of 95% O₂, 5% CO₂ and 15% N₂ maintaining constantpH.

A fraction of the perfusate is diverted to a secondary circuit through ahollow fiber dialyzer, such as Spectrum Labs (Rancho Dominguez, Calif.)with up to 790 cm² membrane area and a 30 kDa molecular weight cutoff(the total filtration surface area in a human kidney is only 516.1 cm²).A particular advantage of the dialysis of the present embodiments isthat albumin, with a molecular weight of 66.5 kDa, is retained in themain perfusion circuit, as further detailed hereinabove.

The secondary circuit dialyzes the perfusate using counter-flow tomaximize diffusion, against a protein-free dialysate, recirculatedthrough an ammonia filter using another peristaltic pump. Ammoniafilters such as Zeolites trap clearing the ammonia from the solution.Temperature within the bioreactor are optimized between 38° C. to 40.5°C. mimicking the normal body temperature of chickens.

Perfusion and nutrient consumption rates are also considered. Underambient conditions, partial pressure of 21% (160 mmHg) of oxygen resultsin a concentration of 220 nmol O₂/ml medium. Using a SeaHorseBioanalyzer the Inventor showed that chicken embryonic fibroblastsconsume 2.4 nmol O₂/min/10⁶ cells. Considering that 1 g of tissuecontains approximately 200×10⁶ cells, a perfusion rate of about 36.9ml/s is sufficient to sustain 1 kg of tissue in standard incubators gaspressures. If oxygen partial pressure is raised to 95%, a perfusion rateof about 8.2 ml/s is sufficient.

Glucose consumption is additionally considered. Using online sensors, aglucose uptake rate of 1.8 nmol/min/10⁶ cells, and lactate productionrate of 198 pmol/min/10⁶ cells were measured for chicken embryonicfibroblasts. Considering that DMEM/F12 medium contains 3.15 g/L ofglucose, the perfusion rate required to sustain 1 kg of tissue would be0.36 ml/sec. Therefore, glucose is not a limiting factor for mediumperfusion. Yet, continuous addition of glucose is preferred, optionallyat narrowing intervals, during late stage culture, since at this rate 1kg tissue consumes the glucose in 1 liter medium within about 48 min.

FIGS. 10A and 10B are graph showing the produce mass and appliedperfusion rates (FIG. 10A), and accumulated glucose consumption (FIG.10B). In this example, an exponential growth rate characterized by adoubling time constant of 20 h has been employed. At this growth rate,it is preferred to add glucose starting from the day 13, so as toprovide the glucose demand.

The peristaltic pumps are selected to provide perfusion rate of at least36 ml/s, particularly towards the last days of the cycle (e.g.,beginning of the 13th day). Yet, this rate can be decreased usingdifferent strategies such as oxygen transporters to increase basal levelof O₂ in the media. For example, Hemopure® is a hemoglobin-based oxygencarrier manufactured by HbO₂ Therapeutics LLC that has an oxygencarrying capacity of 1.39 ml O₂/g Hb, meaning that adding 3.55 μg ofHemopure per ml of media the oxygen content can be doubled, and theperfusion rate can be decreasing by a factor of 2.

Table 3 below provides a comparison between the fed-batch process, theconcentrated perfusion process, and the technique according to exemplaryembodiments of the invention.

TABLE 3 Circulating The inventive Parameter Fed-Batch Perfusiontechnique Seed Train 20 L, 80 L, 20 L run for 10 None 400 L, 2000 Ldays; 6 reactor volumes Production Reactor 10,000 L 1,000 L 5 L CellDensity 25 × 10⁶ 100 × 10⁶ 100 × 10⁶ Growth Phase 19 days 30 days 10days Media Consumption 12,500 L 2,120 L 2 L Media Cost $20/L $5/L $5/LConsumable Costs +$200/kg +$21/kg +$4/kg Facility Cost $50M $30M $5M *Capital Burden $5M $3M $0.5M Production Capacity 24,000 kg/yr 6,000kg/yr 450,000 kg/yr Capital Costs +$200/kg +$500/kg +$1/kg * 5,000 small5 L bioreactors in a factory

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

It will be evident to those skilled in the art that the invention is notlimited to the details of the foregoing illustrated embodiments and thatthe present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

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What is claimed is:
 1. A method of generating a composition comprising acultured fat and a plant-derived protein matrix, the method comprising:culturing an adipocyte cell in vitro to obtain the cultured fat; andadding the plant-derived protein matrix to the cultured fat, therebygenerating the composition comprising cultured fat and a plant-derivedprotein matrix.
 2. The method according to claim 1, wherein theadipocyte cell is generated from a fibroblast.
 3. The method of claim 2,wherein the fibroblast is a spontaneously immortalized fibroblast. 4.The method according to claim 1, wherein the culturing of the adipocytecell occurs in a suspension culture.
 5. The method of according to claim1, wherein the culturing of the adipocyte cell is performed on aplant-derived protein matrix, thereby generating the cultured fat on theplant-derived protein matrix.
 6. The method according to claim 3,further comprising generating the adipocyte cell from a spontaneouslyimmortalized fibroblast by culturing a spontaneously immortalizedfibroblast in a serum-free medium comprising oleic acid and a peroxisomeproliferator-activated receptor gamma (PPAR-gamma) agonist or activator.7. The method of claim 6, wherein the PPAR-gamma agonist or activator isrosiglitazone.
 8. The method of claim 6, wherein the spontaneouslyimmortalized fibroblast is a chicken embryonic fibroblast.
 9. The methodaccording to claim 1, wherein the plant-derived protein matrix comprisesat least one plant-derived protein from the legume (Fabaceae) family,the cereal family, or the pseudocereal family.
 10. The method accordingto claim 9, wherein the at least one plant-derived protein is from thelegume (Fabaceae) family and is selected from the group consisting ofalfalfa, pea, bean, lentil, carob, soybean, and peanut proteins andcombinations thereof.
 11. The method according to claim 10, wherein theat least one plant-derived protein is a soy protein or a pea protein.12. The method according to claim 9, wherein the at least oneplant-derived protein is from the cereal family and is selected from thegroup consisting of maize, rice, wheat, barely, sorghum, millet, oats,rye, tritcale, and fonio proteins and combinations thereof.
 13. Themethod according to claim 9, wherein the at least one plant-derivedprotein is from the pseudocereal family and is selected from the groupconsisting of buckwheat, quinoa, and chia proteins and combinationsthereof.
 14. The method according to claim 1, wherein the plant-derivedprotein matrix comprises a soy protein or a pea protein.
 15. Acomposition produced according to the method of claim 1, wherein thecomposition is a meat substitute product.
 16. A meat substitute productcomprising: a cultured fat obtained from culturing, in-vitro, anadipocyte cell; and a plant-derived protein matrix.
 17. The meatsubstitute product of claim 16, wherein the adipocyte cell is generatedfrom a fibroblast.
 18. The meat substitute product of claim 17, whereinthe fibroblast is a spontaneously immortalized fibroblast.
 19. The meatsubstitute product of claim 16, wherein the adipocyte cell is culturedin a suspension medium.
 20. The meat substitute product of claim 19,wherein the suspension medium is a serum-free medium comprising oleicacid and a peroxisome proliferator-activated receptor gamma (PPAR-gamma)agonist or activator.
 21. The meat substitute product of claim 16,wherein the plant-derived protein matrix comprises at least oneplant-derived protein from the legume (Fabaceae) family, the cerealfamily, or the pseudocereal family.
 22. The meat substitute product ofclaim 21, wherein the at least one plant-derived protein is from thelegume (Fabaceae) family and is selected from the group consisting ofalfalfa, pea, bean, lentils, carob, soybean, and peanut proteins andcombinations thereof.
 23. The meat substitute product of claim 22,wherein the at least one plant-derived protein is a soy protein or a peaprotein.
 24. The meat substitute product of claim 21, wherein the atleast one plant-derived protein is from cereal family and is selectedfrom the group consisting of maize, rice, wheat, barely, sorghum,millet, oats, rye, tritcale, and fonio proteins and combinationsthereof.
 25. The meat substitute product of claim 22, wherein the atleast one plant-derived protein is from the pseudocereal family and isselected from the group consisting of buckwheat, quinoa, and chiaproteins and combinations thereof.
 26. The meat substitute product ofclaim 16, wherein the plant-derived protein matrix comprises a soyprotein or a pea protein.
 27. The meat substitute product of claim 16,wherein the spontaneously immortalized fibroblast is a chicken embryonicfibroblast.
 28. A composition for the in-vitro production of culturedfat, the composition comprising an adipocyte cell; and a serum-freemedium, wherein the adipocyte cell generates the cultured fat.
 29. Thecomposition of claim 28, wherein the adipocyte cell is generated from aspontaneously immortalized fibroblast.
 30. The composition of claim 29,wherein the serum free medium comprises oleic acid and PPAR-gammaagonist or activator.